Natriuretic peptide related fragment in cardiovascular disease

ABSTRACT

This disclosure provides an intracellular fragment of natriuretic peptide receptor A (NPRA), referred to herein as soluble natriuretic peptide receptor-related fragment (sNRF). It is shown herein that sNRF causes NP resistance. Based on these observations, methods of treating a cardiovascular disorder by inhibiting the activity of sNRF are disclosed. Assays are provided that use sNRF to screen agents for their ability to increase the biological activity of an NPR, for example agents that increase the sensitivity of NPR for NPs (such as atrial natriuretic peptide, ANP), or that decrease growth factor deleterious effects, or combinations thereof. Also provided are agents identified using the disclosed assays, and methods of using the agents, for example to treat or diagnose a cardiovascular disorder, such as heart failure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/096,581filed Oct. 15, 2008, now U.S. Pat. No. 7,943,296, which is the U.S.National Stage of International Application No. PCT/US2006/042356, filedOct. 30, 2006, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Patent Application No.60/749,198 filed Dec. 9, 2005, all herein incorporated by reference.

FIELD

This application relates to methods of treating and diagnosing acardiovascular disorder, such as heart failure, methods of identifyingagents that can increase the biological activity of a natriureticpeptide receptor (such as NPRA or NPRB) or can inhibit biologicalactivity of natriuretic peptide-related fragments (such as sNRF), agentsidentified using the method, and methods of using the agents to treat acardiovascular disorder.

BACKGROUND

The proliferation and differentiation of cardiac fibroblasts (CFs) arecritical for the heart's adaptation to pathological stresses (Heling etal., Circ. Res. 86:846-53, 2000). Specifically, CF activity immediatelyafter myocardial infarction (Cameron et al., Endocrinol. 141:4690-7,2000) and during cardiac remodeling (Katz, J. Cell. Mol. Med. 7:1-10,2003; Brown et al., Annu. Rev. Pharmacol. Toxicol. 45:657-87, 2005)leads to myocardial fibrosis and the elaboration of collagen andextracellular matrix, the degree of which largely determines the outcomeof clinical heart failure (Brown et al., Annu. Rev. Pharmacol. Toxicol.45:657-87, 2005; Bax et al., Circulation. 110:1118-22, 2004).

Atrial (ANP) and brain (BNP) natriuretic peptide (NPs) are produced inthe heart and potently inhibit CFs through their ability to bind andactivate the ubiquitous NP receptor, known as natriuretic peptidereceptor A or NPRA (Silberbach and Roberts, Cell. Signal. 13:221-31,2001). Thus, the NP-NPRA system serves as an endogenous defense againstmaladaptive cardiac hypertrophy (Molkentin, J. Clin. Invest. 111:1275-7,2003; Silberbach et al., J. Biol. Chem. 274:24858-64, 1999). However, inthe clinical setting of heart failure, receptor resistance limits thesebeneficial downstream effects (Fan et al., Mol. Pharmacol. 67:174-83,2005; Tsutamoto et al., Circ. 87:70-5, 1993; Nakamura et al., Am. HeartJ. 135:414-20, 1998; Kuhn et al., Cardiovasc. Res. 64:308-14, 2004).Monogenetic mouse models mimic the condition of heart failure-inducedNPRA resistance, in which either cardiac-restricted NPRA deletion(Holtwick et al., J. Clin. Invest. 111:1399-407, 2003) or expression ofa dominant-negative NPRA mutant produces load-independent cardiachypertrophy and fibrosis (Patel et al., Am. J. Physiol. Heart Circ.Physiol. 289:H777-84, 2005). However, in vivo, desensitization of theNPRA receptor is not likely due to such mutations. Therefore, models ofthe in vivo situation are needed, for example to identify agents thatare likely to restore function to NPRA in vivo.

NPRA exists as a homodimer prior to ligand binding. Phosphorylated NPRAis active, and decreased phosphorylation causes receptordesensitization. However, a specific NPRA kinase has not been identified(Potter and Hunter, J. Biol. Chem. 276:6057-60, 2001). NP binding to theextracellular domain is thought to induce a conformational change in thereceptor that results in the juxtaposition of the C-terminal guanylylcyclase domains of the respective NPRA monomers, leading to thegeneration of 5′-cyclic-guanosine-monophosphate (cGMP). cGMP serves as asecond messenger that activates cGMP-dependent protein kinase I (PKG).PKG mediates many NP downstream effects such as cardiac hypertrophy andfibrosis (Silberbach and Roberts, Cell. Signal. 13:221-31, 2001) andpromotes cardiomyocyte survival (Kato et al., J. Clin. Invest.115:2716-30, 2005).

While searching for distal PKG binding partners, an association betweenPKG and a C-terminal fragment of NPRA (NPRA⁽⁸²⁰⁻¹⁰⁶¹⁾) was identified(Airhart et al., J. Biol. Chem. 278:38693-8, 2003). PKG I is a cytosolicserine-threonine kinase that is expressed in a variety of tissues,including the heart and peripheral vasculature. Small quantities ofmembrane-associated PKG I in NPRA over-expressing HEK 293 cells(HEK-NPRA cells), which increased significantly following NP treatment.

Although administration of recombinant NPs has been recently approved bythe FDA, use of such compounds is limited due to NPRA resistance, whichalways occurs in heart failure. In addition, the use of such recombinantNPs may have deleterious long-term effects that lead to kidney failureand increased hospital mortality. Therefore, there is a need to identifyadditional compounds that can be used to treat cardiovascular diseases,such as heart failure.

SUMMARY

In heart failure, the beneficial effects of natriuretic peptides (NPs),including inhibition of growth factor-induced cardiac fibrosis, areblunted. Dysregulation of the NP system is, in part, due to NP receptor(NPRA) unresponsiveness. The inventors have identified an intracellularfragment of natriuretic peptide receptor A (NPRA), called solublenatriuretic peptide receptor-related fragment (sNRF), which appears tocause NP resistance. The sNRF mRNA is the result of transcriptioninitiation in exon 15 of the NPRA gene on human chromosome 1, andencodes a cytosolic protein comprised of more than half of theintracellular portion of NPRA. It is demonstrated herein that sNRFregulates NPRA activation and inhibits NPs' ability to reverse theharmful cardiac effects of growth factors, such as fibroblast growthfactor (FGF) and transforming growth factor-β (TGF-β1).

Based on these observations, methods of treating a cardiovasculardisorder, such as heart failure, by decreasing or inhibiting thebiological activity of sNRF are disclosed. In particular examples, suchmethods can include administration of a therapeutically effective amountof an agent that substantially decreases expression of sNRF, such as aninhibitory RNA molecule. One skilled in the art will appreciate thatcomplete inhibition of sNRF biological activity is not required, asdecreases that have beneficial effects on one or more symptoms of acardiovascular disorder (such as heart failure) are sufficient.

Methods are disclosed for using sNRF (for example when expressed in acardiac cell) to screen for agents that increase the biological activityof NPRA/B, decreased the deleterious cardiac effects of growth factors,or combinations thereof, for example by increasing the sensitivity ofNPRA/B to NP ligand. In particular examples, the method includescontacting a cell (such as a cardiac fibroblast (CF)) with one or moretest agents, a growth factor, and NP (such as ANP), and determiningwhether the test agent increases biological activity of NPRA/B,decreases the deleterious growth factor effects, or both. Such agentscan be used to treat, such as inhibit or prevent, cardiovasculardisease. In particular examples, such agents can also be used todiagnose or determine the severity of a cardiovascular disease.

The cell used in the assay, such as a CF cell, includes a molecule thatcan provide a signal indicating the presence or absence of NPRA/Bbiological activity (such as a promoter operably linked to a reporternucleic acid sequence or a cyclic nucleotide-gated (CNG) channel), NPR(for example a native or recombinant (or both) NPRA or NPRB), and a sNRFthat interferes with binding of cGMP-dependent kinase I (PKG) to NPR. Inparticular examples, the promoter operably linked to a reporter nucleicacid sequence is responsive to one or more growth factors whosedownstream effects are modulated by NP. For example, the deleteriousbiological activities of a growth factor can be decreased in thepresence of NP.

A high throughput approach can be used to screen molecules (such aspeptides, RNAi, or other small molecules) for their ability tocounteract inhibitory effect due to the presence of sNRF that interfereswith binding of PKG to NPR (such as SEQ ID NO: 4, 6, 38, 40 or 42),interferes with other biological actions of sNRF (such as sNRFs abilityto enhance the activity of deleterious growth factor molecules, forexample TGFβ1), or combinations thereof. The molecules that areidentified by such a screen are candidates for drugs that promotePKG-NPRA association and phosphorylation of NPRA and enhance thebeneficial effects of the NP system, drugs that inhibit the deleteriousbiological activities of a growth factor, or both.

In particular examples determining whether the test agent increasesbiological activity of the NPRA/B or interferes with the biologicalactivity of a growth factor includes detecting a signal (such as afluorescent or chemiluminescent signal) generated from a protein encodedby a reporter nucleic acid sequence. Detection of an alteration in thesignal compared to a reference value (such as the signal present in anabsence of the test agent) indicates that the test agent increasesbiological activity of the NPRA/B or interferes with the biologicalactivity of a growth factor. For example, if in the presence of the testagent the signal is significantly altered (such as decreased) relativeto the signal in the absence of the test agent, this indicates that thetest agent may increase biological activity of NPRA/B or interfere withthe biological activity of a growth factor. In another example,determining whether the test agent increases biological activity of theNPRA/B or interferes with the biological activity of a growth factorincludes detecting a signal (such as a fluorescent or chemiluminescentsignal) that results from cyclic nucleotide-gate (CNG) channel activity,such as detecting the influx of calcium or manganese through thechannel. Detection of an alteration in the signal compared to areference value (such as the signal present in an absence of the testagent) indicates that the test agent increases biological activity ofthe NPRA/B, while detection of no significant alteration in the signalcompared to a reference value (such as a change of less than 5%, such asless than 1% as compared to the signal present in an absence of the testagent) indicates that the test agent decreases deleterious growth factoreffects.

Examples of types of agents that can be identified include thefollowing. First are agents that bind to a molecule that isconformationally similar to the NPR association domain on PKG (NAD).These agents may compete with PKG binding to the expressed fragment andthereby promote PKG interaction with NPR. Some of these agents may serveto reduce or inhibit the action of an endogenous molecule (such as sNRF)that inhibits PKG-NPRA association or alters the sub-cellular locationof PKG. Second are agents that bind to a PKG association domain onNPR(PAD) and thereby enhance NPR activity. In particular examples, theseagents can be selected for further analysis. Third are agents that haveaffinity for both NAD and PAD. These agents may promote the associationof NPR and PKG to a greater extent than they promote the association ofsNRF and PKG. In particular examples, agents identified using thedisclosed methods are selected for further analysis, for example testingin an animal model.

Agents identified as candidates to treat cardiovascular disease wherePKG NPRA/B interaction is dysregulated or growth factor biologicalactions are enhanced can be further analyzed. For example, one or moretest agents can be administered to a laboratory mammal havingcardiovascular disease where NPRA/B-PKG association is dysregulated, andthen determining whether the test agent treats the cardiovasculardisease. Alternatively, one or more test agents can be administered to alaboratory mammal having cardiovascular disease because growth factoraction is enhanced, and then determining whether the test agent treatsthe cardiovascular disease.

Also provided by the present disclosure are agents identified using thedisclosed assays.

Methods are provided of treating a subject having cardiovascular disease(or having an increased risk of developing cardiovascular disease), forexample by administration of a therapeutically effective amount of oneor more therapeutic agents identified using the disclosed methods.

Also disclosed are methods of using compounds identified using themethods to diagnose cardiovascular disease in a subject. Individualswith latent or subclinical cardiovascular disease would respond to atherapeutically effective amount of one or more therapeutic agentsidentified using the disclosed methods. Those not responding would nothave dysregulation of NPR-PKG association or do not have increaseddeleterious growth factor actions.

Also provided are methods of using sNRF to diagnose a cardiovasculardisorder, for example to determine the severity of heart failure.

The present disclosure also provides sNRF protein and nucleic acidsequences. For example, exemplary sNRF protein sequences are shown inSEQ ID NOS: 4, 6, 8, 38, 40 and 42, and exemplary sNRF nucleic acidsequences are shown in SEQ ID NOS: 3, 5, 7, 37, 39, and 41.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing that prolonged NP stimulation of CF cellscauses NP resistance. Data represents mean±SE for 4 independentexperiments, each performed in duplicate. *difference in luciferaseexpression between TGF-β₁ and TGF-β₁+NP compared to day 1, p<0.05.

FIG. 2 is a schematic drawing showing the proposed sNRF transcriptionpattern. Forward primer “a” corresponds to intron 15 sequence andreverse primer “b” skips intron 16 and is complementary to exon 17sequence. RT-PCR with primers “a” and “b” produces a 387-bp product.These primers can be employed to identify sNRF expression specificallyand will not recognize NPRA because NPRA mRNA contains no intron 15sequence.

FIG. 3 is a digital image of a Northern blot containing total mRNAextracted from human heart hybridized with a probe to the 215-ntintron-15 sequence of NPRA. A prominent band can be seen atapproximately 1.5 kb.

FIG. 4A is a bar graph showing the relative expression of sNRF inpoly(A)⁺ mRNA extracted from an explanted heart from a patient withdilated cardiomyopathy (DCM) compared to poly(A)⁺ mRNA extracted from apost-mortem specimen from a patient who died from a non-cardiac cause.

FIG. 4B is a bar graph showing that sNRF and full-length NPRA aredifferentially regulated in heart failure. Upper panel shows therelative expression of total sNRF mRNA extracted from the explantedheart of a patient with hypertrophic cardiomyopathy compared to “normal”heart total mRNA. Lower panel shows the relative expression offull-length mRNA using a Taqman probe targeted to exon 7 and 8 exonicsequences that are not present in the sNRF mRNA.

FIG. 4C shows the results of quantitative RT-PCR of total RNA usingeither a sNRF-specific or NPRA-specific probe on each of three explantedfailing hearts (patients #3, 4, 5). Values represent fold changerelative to the lowest value.

FIG. 4D is a bar graph showing the variable sNRF expression in childhoodheart failure. Data are fold change relative to the first heart.(CHD=congenital heart disease, DCM=dilated cardiomyopathy,HCM=hypertrophic cardiomyopathy, RCM=restrictive cardiomyopathy).

FIGS. 5A and 5B are bar graphs showing the relative amount of (A) sNRFand (B) NPRA mRNA expression in normal hearts or heart failure hearts.

FIG. 6A is a digital image of a Western blot showing that PKGphosphorylates NPRA.

FIG. 6B is a digital image of a Western blot showing NP-dependentphosphorylation of NPRA in HEK293 that overexpress NPRA. Phosphorylationis inhibited by the PKG inhibitor KT5823. No phosphorylation is observedin control cells that do not express NPRA.

FIGS. 6C and 6D are (C) a digital image of a Western blot showing in CFcells expressing endogenous NPRA, NP-dependent phosphorylation, and (D)quantification of the signals in FIG. 6C by scanning densitometry (dataare the mean±SEM of 4 identical experiments). NP dependentphosphorylation is inhibited by the PKG inhibitor KT5823 andsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾.

FIG. 7 is a bar graph showing that sNRF mimics NP receptorunresponsiveness observed in heart failure.

FIG. 8A is a digital image of a Western immunoblot showing thatsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ reverses ANP inhibition of FGF-induced α-SMA expression.Representative Western immunoblot probed with anti-α-SMA antibody,stripped and re-probed with anti-vimentin antibody.

FIG. 8B is a bar graph showing the quantification of the signals fromFIG. 8A obtained by scanning densitometry and normalized to vimentin.Data are the mean±SE of 6 identical experiments.

FIG. 9 is a bar graph showing that sNRF inhibits NP action andangiotensin-inhibiting drugs do not block sNRF effects. Data is mean±SEMand represents 6 separate experiments (except for LOS n=2).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NOS: 1 and 2 show an exemplary full-length NPRA mRNA and proteinsequence, respectively (GenBank Accession No. NM_(—)000906).

SEQ ID NOS: 3-6 show exemplary intracellular NPRA fragments that canbind PKG (SEQ ID NOs: 4 and 6) and the corresponding mRNA sequences (SEQID NO: 3 and 5).

SEQ ID NOS: 7 and 8 show an exemplary NPRA fragment (sNRF⁽⁸⁰⁶⁻⁸⁶⁰⁾, SEQID NO: 8) that only includes the NPRA hinge domain, and does notspecifically bind PKG. The corresponding mRNA sequence is shown in SEQID NO: 7.

SEQ ID NOS: 9 and 10 show primers that can be used to generate a 5′-FLAGepitope-tagged sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾ fragment.

SEQ ID NOS: 9 and 11 show primers that can be used to generate a 5′-FLAGepitope-tagged sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ fragment.

SEQ ID NOS: 9 and 12 show primers that can be used to generate a 5′-FLAGepitope-tagged sNRF⁽⁸⁰⁶⁻⁸⁶⁰⁾ fragment.

SEQ ID NOS: 13 and 14 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 15 and 16 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 17 and 18 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 19 and 20 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 21 and 22 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 23 and 24 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 25 and 26 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 27 and 28 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 29 and 30 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 31 and 32 show an exemplary sNRF coding sequence and thecorresponding siRNA sequence, respectively, that can be used to inhibitsNRF expression.

SEQ ID NOS: 33 and 34 show forward and reverse primers, respectively,that can be used to amplify sNRF using PCR (primers “a” and “b” in FIG.2).

SEQ ID NO: 35 shows an NPRA intron 15-specific probe that can be usedfor Northern blotting of RNA.

SEQ ID NO: 36 shows a TaqMan probe containing a 5′ label (FAM) and a 3′label (TAMRA) that can be used to quantitate sNRF expression usingreal-time PCR.

SEQ ID NO: 37 shows a partial genomic sequence of an exemplary sNRF,that includes intron 15 (nucleotides 1-215) and a partial codingsequence (nucleotides 256-816).

SEQ ID NO: 38 shows the sequence encoded by SEQ ID NO: 37, and providesan exemplary sNRF protein.

SEQ ID NOS: 39-40 show an exemplary sNRF (sNRF⁽⁸²⁰⁻¹⁰⁶¹⁾) that can bindPKG (SEQ ID NO: 40) and the corresponding cDNA sequence (SEQ ID NO: 39).

SEQ ID NOS: 41-42 show an exemplary sNRF (sNRF⁽⁸²⁰⁻⁹⁰⁰⁾) that can bindPKG (SEQ ID NO: 42) and the corresponding cDNA sequence (SEQ ID NO: 41).

SEQ ID NOS: 43 and 44 show forward and reverse primers, respectively,that can be used to amplify sNRF using quantitative real-time PCR(primers “a” and “b” in FIG. 2).

DETAILED DESCRIPTION Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein,“comprising” means “including” and the singular forms “a” or “an” or“the” include plural references unless the context clearly dictatesotherwise. For example, reference to “comprising a cell” includes one ora plurality of such cells, and reference to “comprising a test agent”includes reference to one or more test agents and equivalents thereofknown to those skilled in the art, and so forth. The term “or” refers toa single element of stated alternative elements or a combination of twoor more elements, unless the context clearly indicates otherwise. Forexample, the phrase “A or B” refers to A, B, or a combination of both Aand B.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the disclosure are apparent from the following detaileddescription and the claims.

ANP atrial natriuretic peptide

α-SMA alpha smooth muscle actin

CF cardiac fibroblast

CNG channel cyclic nucleotide-gated channel

NP natriuretic peptide

NPRA natriuretic peptide receptor A

NPRA/B natriuretic peptide receptor A, NPRB, or both

PKG cGMP-dependent protein kinase I

siRNA short interfering or interrupting RNA

sNRF soluble natriuretic peptide receptor-related fragment

Administer: To provide or give a subject an agent, such as compositionthat includes the agent, by any effective route. Exemplary routes ofadministration include, but are not limited to, oral, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal, intranasal, andinhalation routes. In particular examples, agents (such as thoseidentified using the disclosed methods) are administered to a subjecthaving cardiovascular disease or having an increased risk for developingcardiovascular disease. In one example, one or more agents thatsubstantially decrease the biological activity of sNRF (for exampledecrease sNRF expression), is administered to a subject havingcardiovascular disease or having an increased risk for developingcardiovascular disease.

Alpha smooth muscle actin (α-SMA) promoter: A nucleic acid sequence thatcan promote the expression of α-SMA (an actin isoform that is a markerfor myofibroblasts in the diseased heart) in vivo. In addition, an α-SMApromoter sequence can drive the expression of a nucleic acid sequenceoperably linked to it in the presence of a growth factor in vitro.

Includes any α-SMA promoter nucleic acid molecule from any organism,such as a mammal. This description includes α-SMA promoter variants thatretain the ability to drive the expression of a nucleic acid sequenceoperably linked downstream of the promoter in the presence of a growthfactor. For example, α-SMA promoters can promote the expression of areporter nucleic acid sequence operably linked to the promoter, in thepresence of a growth factor (such as TGF-β).

Nucleic acid α-SMA promoter sequences are publicly available. Oneparticular example of an α-SMA promoter that can be used is disclosed inWang et al. (J. Clin. Invest. 100:1425-39, 1997) (the mouse SMC α-actinpromoter fragment SMP8 (−1074 bp, 63 bp of 5′UT and 2.5 kb of intron 1).

Antisense, Sense, and Antigene: Antisense molecules are molecules thatare specifically hybridizable or specifically complementary to eitherRNA or the plus strand of DNA. Sense molecules are molecules that arespecifically hybridizable or specifically complementary to the minusstrand of DNA. Antigene molecules are either antisense or sensemolecules directed to a particular dsDNA target (such as sNRF, forexample intron 15 of NPRA). These molecules can be used to interferewith gene expression, such as expression of sNRF.

Binding: A specific interaction between two or more molecules, such thatthe two or more molecules interact. Therefore to “interfere withbinding” refers to disrupting this interaction, for example a disruptionof at least 50%, at least 75%, or at least 90%.

For example, binding can occur between a NPR and PKG, and between NPR(such as NPRA or NPRB) and a particular NP ligand, such as ANP or BNP.The binding is a non-random binding reaction, for example between twoproteins. Binding can be specific and selective, so that one molecule isbound preferentially when compared to another molecule. Bindingspecificity of one agent for another agent is typically determined fromthe reference point of the ability of the agent to differentially bind aspecific agent and an unrelated agent, and therefore distinguish betweentwo different agents.

In particular examples, two compounds are said to specifically bind whenthe binding constant for complex formation between the componentsexceeds about 10⁴ L/mol, for example, exceeds about 10⁶ L/mol, exceedsabout 10⁸ L/mol, or exceeds about 10¹⁰ L/mol. The binding constant fortwo components can be determined using methods that are well known inthe art.

Cardiac fibroblast (CF): A fibroblast of the heart that can produceextracellular matrix proteins (such as collagen). CFs can be obtainedfrom a primary culture (for example using the method of Simpson,Circulation Res. 56:884-94, 1985), or can be obtained from a commercialsource, for example Cell Applications, Inc., San Diego, Calif. (human orrat CF cells derived from normal heart tissue).

Cardiovascular disease: Any disorder that affects the heart or thevasculature. Particular conditions include, but are not limited to:angina pectoris; arrhythmia; cardiac fibrosis; congenital cardiovasculardisease; coronary artery disease (CAD); dilated cardiomyopathy; heartattack (myocardial infarction); heart failure; hypertrophiccardiomyopathy; systemic hypertension from any cause; edematousdisorders caused by liver or renal disease; mitral regurgitation;myocardial tumors; myocarditis; rheumatic fever; Kawasaki disease;Takaysu arteritis; cor pulmonale; primary pulmonary hypertension;amyloidosis; hemachromatosis; toxic effects on the heart due topoisoning; Chaga's disease; heart transplantation; cardiac rejectionafter heart transplantation; cardiomyopathy of chachexia; arrhythmogenicright ventricular dysplasia; cardiomyopathy of pregnancy; MarfanSyndrome; Turner Syndrome; Loeys-Dietz Syndrome; familial biscuspidaortic valve or any inherited disorder of the heart or vasculature, orcombinations thereof.

In some examples, cardiovascular disease is caused by a decrease in thebiological activity of NPRA, for example a decrease in the sensitivityof NPRA for NP ligands such as ANP. In some examples, cardiovasculardisease is caused by an increase in the biological activity of a growthfactor (such as FGF or TGFβ1), for example in situations where growthfactor action is enhanced by the biological activity of sNRF.

In particular examples, cardiovascular disease is treated byadministration of one or more of the agents identified using the methodsdisclosed herein. In one example, cardiovascular disease is treated byadministration of one or more agents that substantially decrease thebiological activity of sNRF (for example decrease sNRF expression).

Conservative substitution: One or more amino acid substitutions (forexample 1, 2, 5, 8, or 10 amino acid residues) for amino acid residueshaving similar biochemical properties. Typically, conservativesubstitutions have little to no impact on the activity of a resultingpeptide. For example, a conservative substitution is an amino acidsubstitution in an intracellular NPRA peptide fragment, such as sNRF,that does not substantially affect the ability of the peptide to bind toPKG. In a particular example, a conservative substitution is an aminoacid substitution in a sNRF, such as a conservative substitution in SEQID NO: 4, 6, 38, 40 or 42 that does not significantly alter the abilityof the peptide to bind to NPRA or the ability of the peptide to reduceNPs' inhibitory effect on deleterious growth factor effects.

An alanine scan can be used to identify amino acid residues in a peptide(such as a sNRF, such as SEQ ID NO: 4, 6, 38, 40 or 42) that cantolerate substitution. In one example, activity is not altered by morethan 25%, for example not more than 20%, for example not more than 10%,when an alanine, or other conservative amino acid (such as those listedbelow), is substituted for one or more native amino acids.

In a particular example, the activity of a sNRF is not substantiallyaltered if the amount of cGMP produced by NPRA in the presence of thesNRF peptide fragment is not reduced by more than about 25%, such as notmore than about 10%, than an amount of cGMP produced by NPRA in thepresence of the sNRF containing one or more conservative amino acidsubstitutions, as compared to an amount of cGMP production in thepresence of a native sNRF sequence.

A peptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpeptide using, for example, standard procedures such as site-directedmutagenesis or PCR. Alternatively, a peptide can be produced to containone or more conservative substitutions by using standard peptidesynthesis methods.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Examples of amino acids which may be substituted for anoriginal amino acid in a protein and which are regarded as conservativesubstitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glufor Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Glnfor His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leuor Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyrfor Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

Further information about conservative substitutions can be found in,among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988), WO 00/67796 (Curd et al.) and in standard textbooks of geneticsand molecular biology.

Decrease: To reduce the quality, amount, or strength of something.

In one example, a therapy decreases cardiovascular disease (such ascardiac fibrosis, myocyte apoptosis or expression of inflammatorycytokines), or one or more symptoms associated with cardiovasculardisease (such as decreased urine output, decreased ability to exercise,dyspnea, decrease peripheral edema due to fluid retention, and all othersigns or symptoms of heart failure known by skilled clinicians), forexample as compared to the response in the absence of the therapy. In aparticular example, a therapy decreases cardiovascular disease or asymptom of cardiovascular disease, subsequent to the therapy, such as adecrease of at least 10%, at least 20%, at least 50%, or even at least90%. Such decreases can be measured using routine clinical methods.Examples of such therapies include administration of therapeuticallyeffective amounts of agents identified using the methods disclosedherein or administration of therapeutically effective amounts of agentsthat substantially decrease the biological activity of sNRF (such as asNRF siRNA molecule).

Decrease activity: An agent is said to “decrease activity” of a sNRFwhen contact of the agent results in decreased biological activity ofthe sNRF as compared to an amount of biological activity of sNRF notpreviously contacted with the agent. Decreasing the activity of a sNRFdoes not require complete inhibition of sNRF activity. For example, adecrease in biological activity of at least 25%, such as at least 50%,or at least 75%, when compared to no treatment with the agent indicatesthat the agent can decrease the biological activity of the sNRF.

In one example, decreasing the activity of sNRF increases thesensitivity of the NPRA/B receptor for NPs, such as ANP, as compared tothe sensitivity in the absence of the agent that decreases the activityof sNRF. For example, this increased sensitivity can result in increasedcGMP levels in response to NP treatment. In some examples, binding of NPto NPR having increased sensitivity to NP, increases at least 25% whencompared to sensitivity in the absence of the agent, such as increasesof at least 50%, at least 75%, at least 80%, at least 90%, at least100%, or even at least 200%. Cyclic-GMP levels can be measured in wholecell lysates by enzyme immunoabsorbance assay (EIA) or cGMP cyclaseactivity assays where crude membranes are assayed in the presence of ATPand [α-³²P]GTP (for example as described in Airhart et al., J. Biol.Chem. 278:38693-8, 2003).

In another or in an additional example, an agent is said to decrease theactivity of sNRF when contact of the agent with NPRA/B, or PKG, or bothNPRA/B and PKG results in a decrease in the production of α-SMA asmeasured by Western immunoblot, as compared to production in the absenceof the agent, such as a decrease of at least 25% when compared to theabsence of the agent, such a decrease of at least 50%, at least 60%, atleast 75%, at least 80%, or at least 90%.

In another or in an additional example, an agent is said to decrease theactivity of sNRF when contact of the agent with a cardiofibroblast underconditions that mimic heart failure results in a decrease in thedeleterious growth factor effects (such as proliferation of the cells,and expression of collagen, extracellular matrix, and α-SMA), ascompared to production in the absence of the agent, such as a decreaseof at least 25% when compared to the absence of the agent, such adecrease of at least 50%, at least 60%, at least 75%, at least 80%, orat least 90%.

In a clinical setting an agent is said to decrease the activity of sNRFwhen treatment of a subject with the agent results in the typicalresponse of a patient as if sNRF expression was not increased, forexample as if NPRA/B is not desensitized, if deleterious growth factoreffects were not observed, or combinations thereof. Such a subject mighthave normalized blood pressure, increased urine output, suppression ofnumerous neuroendocrine markers of heart failure such as angiotensin,aldosterone, endothelin, renin, the sympathetic nervous system, or othergrowth factors, combing programmed cell death (apoptosis), decreasedcardiac fibrosis, decrease in cardiac filling pressures, improvement ofcardiac output, lessening of angina pectoris and in general diminutionof the typical signs and symptoms of cardiovascular disease.

Enhance: To improve the quality, amount, or strength of something.

In one example, a therapy enhances the sensitivity of a NPRA/B tobinding by NP ligands (such as ANP or BNP), if the biological activityof NPRA/B increases in the presence of NPs and the therapy, as comparedto the biological activity of NPRA/B in the presence of NPs and absenceof the therapy. In a particular example, a therapy enhances thesensitivity of NPRA/B to binding by NP ligands if the biologicalactivity of NPR increases subsequent to the therapy, such as an increaseof at least 10%, at least 20%, at least 50%, or even at least 90%. Suchenhancement can be measured using the methods disclosed herein, forexample determining an amount of cGMP using an enzyme immunoabsorbance(EIA) assay. Examples of such therapies include administration oftherapeutically effective amounts of agents identified using the methodsdisclosed herein or administration of therapeutically effective amountsof agents that substantially decrease the biological activity of sNRF(such as a sNRF siRNA molecule).

Expression: With respect to a gene sequence, refers to transcription ofthe gene and, as appropriate, translation of the resulting mRNAtranscript to a protein. Thus, expression of a protein coding sequenceresults from transcription and translation of the coding sequence.

Growth factor: Substances that responsible for regulating cellproliferation, development, migration, or differentiation. In someexamples, growth factors include those whose expression is increased inresponse to cardiovascular disease, such as heart failure. For example,a growth factor can be one whose biological activity is modulated (suchexample activated or repressed) in the presence of NPs, such as ANP. Ina particular example, a growth factor is one whose biological activityis decreased in the presence ANP.

Particular examples of growth factors that can be used in the screeningmethods provided herein include, but are not limited to, epidermalgrowth factor (EGF), fibroblast growth factor (FGF), erythropoietin(EPO), growth hormone (GH), insulin-like growth factor, hematopoieticcell growth factor (HCGF), platelet-derived growth factor (PDGF),vascular endothelial growth factor (VEGF), and transforming growthfactors (such as TGF-β)

A deleterious growth factor affect or growth factor deleterious effectsis a negative consequence to a cardiac cell (such as a cardiacfibroblast) in the presence of growth factors, such as FGF and TGFβ1.Examples of such negative consequences are those that can causecardiovascular disease (such as heart failure), for example cellularproliferation, expression of collagen, extracellular matrix, and α-SMA,or combinations thereof. In particular examples, such negativeconsequences can be decreased in the presence of NP, but are negated inthe presence of sNRF.

Heart failure: The condition that results when the cardiac output isinsufficient to meet the metabolic needs of the body. This can occurwhen the heart muscles contract or relax abnormally. With heart failure,the cardiac output may be decreased and the cardiac chamber fillingpressures increase. The chambers of the heart also increase in size inorder to hold more blood to pump through the body. When the cardiacoutput decreases the kidneys often respond by causing the body to retainfluid (water) and sodium. If fluid builds up in the arms, legs, ankles,feet, lungs or other organs, the body's tissues becomes congested(edema). Therefore, heart failure can be characterized by one or more ofthe following symptoms: total body fluid overload, over-activation of avariety of deleterious hormones (such as catecholamines includingneuroepinephine and epinephrine; vasoconstricting hormones angiotensinII and endothelin, inflammatory agents such as cytokines, fibroblastgrowth factor, transforming growth factor beta, and numerous others),and maladaptive thickening of the heart muscle (hypertrophy) thatinvolves cardiac fibrosis. Causes of heart failure include, but are notlimited to, coronary disease, heart attack, non-ischemic cardiomyopathy,as well as conditions that stress the heart (such as high bloodpressure, valve disease, thyroid disease, kidney disease, diabetes orcardiac malformations).

Early heart failure (compensated heart failure) occurs when natriureticpeptides (NPs) and other systems effectively oppose the effects of fluidoverload, hormonal activation, and maladaptive hypertrophy.Decompensated heart failure occurs when NP action is overwhelmed (inspite of the presence of high circulating NP levels), which can resultin death.

In particular examples, heart failure is treated by administration oftherapeutically effective amounts one or more of the agents identifiedusing the methods disclosed herein. In some examples, heart failure istreated by administration of therapeutically effective amounts of agentsthat increase the activity of NPR, substantially decrease the biologicalactivity of sNRF (such as a sNRF siRNA molecule or other chemicalcompounds), decrease growth factor deleterious effects, or combinationsthereof.

Increase activity: An agent is said to “increase activity” of anatriuretic peptide receptor (NPR, such as NPRA/B) when contact of theagent with a desensitized or substantially inactive NPRA/B results inincreased biological activity of the desensitized NPRA/B, as compared toan amount of biological activity of a desensitized NPRA/B not previouslycontacted with the agent. Increasing the activity of a desensitizedNPRA/B does not require restoration of 100% of activity present when thereceptor is sensitized. For example, an increase in biological activityof at least 25% when compared to no treatment with the agent indicatesthat the agent can increase the biological activity of the desensitizedNPRA/B.

In one example, increasing the activity of NPRA/B (such as desensitizedNPRA/B) increases the sensitivity of the receptor for NPs, such as ANP,as compared to the sensitivity in the absence of the agent. For example,this increased sensitivity can result in increased cGMP levels inresponse to NP treatment. In some examples, binding of NP to NPR havingincreased sensitivity to NP, increases at least 25% when compared tosensitivity in the absence of the agent, such as increases of at least50%, at least 75%, at least 80%, at least 90%, at least 100%, or even atleast 200%. Cyclic-GMP levels can be measured in whole cell lysates byenzyme immunoabsorbance assay (EIA) or cGMP cyclase activity assayswhere crude membranes are assayed in the presence of ATP and [α-³²P]GTP(for example as described in Airhart et al., J. Biol. Chem. 278:38693-8,2003).

In another or in an additional example, an agent is said to increase theactivity of NPRA/B (such as desensitized NPRA/B) when contact of theagent with NPRA/B, or PKG, or both NPRA/B and PKG results in a decreasein the production of α-SMA as measured by Western immunoblot, ascompared to production in the absence of the agent. For example,production of an intracellular marker of NPRA/B function (such as cGMPor α-SMA) by an NPRA/B having increased biological activity, canincrease or decrease by at least 25% when compared to production of anintracellular marker of NPRA/B function in the absence of the agent,such as changes of at least 50%, at least 60%, at least 75%, at least80%, or at least 90%. Determining an amount of production of anintracellular marker of NPRA/B function can be performed using themethods disclosed herein.

In a clinical setting an agent is said to increase the activity ofNPRA/B (such as desensitized NPRA/B) when treatment of a subject withthe agent results in the typical response of a patient as if the NPRA/Bis not desensitized. Such a subject might have normalized bloodpressure, increased urine output, suppression of numerous neuroendocrinemarkers of heart failure such as angiotensin, aldosterone, endothelin,renin, the sympathetic nervous system, or other growth factors, combingprogrammed cell death (apoptosis), decreased cardiac fibrosis, decreasein cardiac filling pressures, improvement of cardiac output, lesseningof angina pectoris and in general diminution of the typical signs andsymptoms of cardiovascular disease.

Mammal: Includes both human and non-human mammals. Similarly, the terms“patient,” “subject,” and “individual” includes living multicellularvertebrate organisms, such as human and veterinary subjects.

MicroRNA (miR): A small non coding RNA sequence that directs posttranscriptional regulation of gene expression through interaction with ahomologous mRNA. MiRs can inhibit translation, or can direct cleavage oftarget mRNAs. Therefore, miRs can be used to decrease or inhibitexpression of sNRF, for example to treat heart failure. In particularexamples, miRs are about 21-26 nucleotides in length.

Natriuretic peptide (NP): A family of peptide hormones that regulatemammalian blood volume and blood pressure, and which are naturalantagonists to the renin-angiotensin-aldosterone system. Members includeatrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides. ANPand BNP are released primarily from the heart, while CNP is releasedprimarily from noncardiac tissues (such as the endothelium).

ANP is a 28 amino acid peptide (for example see amino acids 124 to 151of GenBank Accession No. AAA35529) that is synthesized, stored, andreleased by atrial myocytes in response to atrial distension,angiotensin II stimulation, endothelin, and sympathetic stimulation(beta-adrenoceptor mediated). Therefore, elevated levels of ANP arefound during hypervolemic states (elevated blood volume) and congestiveheart failure.

Natriuretic peptide receptor (NPR): A family of receptors thatspecifically bind to NPs. Natriuretic peptide receptor A (NPRA) and B(NPRB) are members of the transmembrane guanylyl cyclase family thatmediate the effects of natriuretic peptides via the second messengercyclic GMP (cGMP). ANP and BNP are extracellular ligands for NPRA, whileCNP is an extracellular ligand for NPRB. When ANP, BNP or CNP bind toNPRA or NPRB, an increase in guanylate cyclase activity results leadingto production of cGMP. NPRC is not linked to guanylyl cyclase and servesas a clearance receptor.

The NPRA and NPRB receptors (NPRA/B) are composed of an extracellularligand binding, transmembrane, protein kinase-like, hinge, and catalyticdomains. The location of such domains is publicly available. Forexample, NPRA and NPRB have an about 450 amino acid extracellular ligandbinding domain, a 21 amino acid hydrophobic membrane-spanning region,and about 566-568 intracellular amino acids (which can be divided into ajuxtamembrane region of about 250 amino acids (kinase homology domain),a 41 amino acid hinge region, and an about 250 amino acid guanylylcyclase catalytic domain). In a particular example, in human NPRA, theextracellular ligand binding domain includes amino acids 54-415, thetransmembrane domain includes amino acids 477-493, the proteinkinase-like domain includes amino acids 547-804, hinge domain includesamino acids 805-848, and the catalytic domain includes amino acids840-1023 (amino acids refer to SEQ ID NO: 2).

NPRs are present in most tissues of the body including cardio-myocytesand fibroblasts. Binding of NP ligands (such as ANP and BNP) to NPRs(such as NPRA) in cells of the heart reduce or inhibit the biologicalactivity of growth factors that are present during heart failure (suchas FGF, EGF, and TGF-β) Therefore, the biological activity of NPRprovides a cardioprotective function, by limiting the hypertrophic andfibrotic response to pressure overload, suppressing the neuroendocrinemileu of heart failure, inhibiting programmed cell death (apoptosis) incardiomyocytes thus potentially reducing mortality. In particularexamples, such activity is achieved upon binding of NPs to theextracellular domain of NPR.

In living subjects NPRA/B function can assessed by measuring thecirculating levels or urinary levels of cGMP in response to NP infusion.A typical bioassay of NPR function is to measure changes in forearmvascular resistance in response to NP infusion. Other common bioassayscan be the urine output response to NP infusion. In the hospital settingfavorable changes in cardiac filling pressure, decrease in the painassociated with angina pectoris and improved sense of well-being can bemeasured.

NPRA and NPRB protein and nucleic acid sequences are publicly available,for example from EMBL or GenBank. For example, GenBank Accession Nos.AAH63304 and AAA66945 provide human and mouse NPRA protein sequences,respectively. GenBank Accession Nos. BC063304 and L31932 provides humanand mouse NPRA nucleic acid sequences, respectively. One particularexample of a full-length NPRA protein sequence is shown in SEQ ID NO: 2.

Nucleic acid molecule: Encompasses both RNA and DNA including, withoutlimitation, cDNA, genomic DNA, mRNA. Includes synthetic nucleic acidmolecules, such as those that are chemically synthesized orrecombinantly produced. The nucleic acid molecule can be double-strandedor single-stranded. Where single-stranded, the nucleic acid molecule canbe the sense strand or the antisense strand. In addition, nucleic acidmolecules can be circular or linear.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence (such as a reporter sequence). Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein coding regions, in the same reading frame.

Peripheral administration: Administration outside of the central nervoussystem. Peripheral administration does not include direct administrationto the brain. Peripheral administration includes, but is not limited tointravascular, intramuscular, subcutaneous, inhalation, oral, rectal,transdermal or intra-nasal administration.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic effect when administered to a subject,alone or in combination with another therapeutic agent(s) orpharmaceutically acceptable carriers. In a particular example, apharmaceutical agent treats heart failure, for example by reducing oneor more signs or symptoms of heart failure.

Promoter: An array of nucleic acid control sequences that directtranscription of a nucleic acid molecule, such as a reporter sequence. Apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription.

The term includes endogenous promoter sequences as well as exogenouspromoter sequences. In one example, the promoter is an induciblepromoter, such as a promoter responsive to a particular stimulus. In aparticular example, the inducible promoter is one that is responsive tothe presence of a growth factor that is responsive to NPs, such asepidermal growth factor (EGF), fibroblast growth factor (FGF), orTGF-β1. Particular examples of promoters that are responsive to a growthfactor that is responsive to NPs include, but are not limited to: alphasmooth muscle actin promoter (α-SMA), pro alpha 2(I) collagen promoter,β-myosin heavy chain promoter, or an atrial natriuretic peptidepromoter.

Recombinant: A recombinant nucleic acid molecule or protein is one thathas a sequence that is not naturally occurring, has a sequence that ismade by an artificial combination of two otherwise separated segments ofsequence, or both. This artificial combination can be achieved, forexample, by chemical synthesis or by the artificial manipulation ofisolated segments of nucleic acid molecules or proteins, such as geneticengineering techniques. Recombinant is also used to describe nucleicacid molecules that have been artificially manipulated, but contain thesame regulatory sequences and coding regions that are found in theorganism from which the nucleic acid molecule was isolated.

Reporter: A molecule that produces a detectable signal when a targetmolecule present. In particular examples, a detectable molecule producesa colorimetric signal, for example a luminescent or fluorescent signal.Methods of detecting such signals are known in the art, and can include,but are not limited to, ELISA, spectrophotometry, flow cytometry, ormicroscopy.

In particular examples, a reporter is a nucleic acid sequence thatproduces a detectable protein when expressed. A reporter nucleic acidmolecule can include a promoter, the structural sequence of the reportergene, and the sequences required for the formation of functional mRNA.Upon introduction of the reporter construct into cells, expressionlevels of the reporter gene can be monitored, for example by assayingfor the reporter protein's enzymatic activity, or by measuringproduction of the protein directly (for example if the protein is afluorescent or luminescent protein, such as green fluorescent protein(GFP), fluorescence or luminescence can be detected). Particularexamples include, but are not limited to: luciferase, β-galactosidase,chloramphenicol acetyltransferase (CAT), GFP (or variants thereof suchas E-GFP). Sequences for such reporter molecules are well-known in theart. For example, a promoter (such as one that is responsive to growthfactors that are modulated by NPs) can be inserted into these plasmidsthat include the indicated reporter: p-lacZ (beta-galactosidasereporter), p-luc (firefly luciferase reporter) and p-cat(chloramphenicol acetyl transferase).

In other particular examples, a reporter is molecule that can detect thepresence of another molecule, such the ability of fura-2 to detect thepresence of Ca²⁺ or Mn²⁺ influx through a CNG channel. For example, ANPcan increase cGMP levels, thereby activating CNG channels, as detectedby the presence Ca²⁺ or Mn²⁺ by measuring fura-2 levels.

RNA interference (RNAi): A post-transcriptional gene silencing mechanismmediated by double-stranded RNA (dsRNA). Introduction of dsRNA intocells, such as siRNA compounds, induces targeted degradation of RNAmolecules with homologous sequences. RNAi compounds can be used tomodulate transcription, for example, by silencing genes, such as sNRF(for example by targeting nucleotides 1-215 of SEQ ID NO: 37). Incertain examples, an RNAi molecule is directed against a target, such assNRF, and is used to treat heart failure.

Short interfering or interrupting RNA (siRNA): Double-stranded RNAs thatcan induce sequence-specific post-transcriptional gene silencing,thereby decreasing or even inhibiting gene expression. In some examples,siRNA molecules are about 19-23 nucleotides (nt) in length, such as21-23 nt. In particular examples, siRNA molecules are at least 21 nt,for example at least 23 nt in length. In one example, an siRNA moleculeis an siRNA duplex composed of 21-nt sense and 21-nt antisense strands,paired in a manner to have a 2-nt 3′ overhang on both strands. Inanother example, an siRNA is 19 nt in length having two dT overhangs atthe N- and C-terminal ends. In a particular example, an siRNA moleculeselectively binds to intron 15 of NPRA, thereby decreasing expression ofsNRF.

In one example, siRNA triggers the specific degradation of homologousRNA molecules, such as mRNAs, within the region of sequence identitybetween both the siRNA and the target RNA (such as sNRF RNA). Forexample, WO 02/44321 discloses siRNAs capable of sequence-specificdegradation of target mRNAs when base-paired with 3′ overhanging ends.The direction of dsRNA processing determines whether a sense or anantisense target RNA can be cleaved by the produced siRNA endonucleasecomplex. Thus, siRNAs can be used to modulate transcription, forexample, by silencing genes, such as sNRF, for example to treat heartfailure. Particular exemplary siRNA molecules that can be used tosilence sNRF expression include those shown in SEQ ID NOS: 14, 16, 18,20, 22, 24, 26, 28, 30, and 32.

Signal: A detectable change in a physical quantity or impulse thatprovides information. In the context of the disclosed methods, examplesinclude light, such as light of a particular quantity or wavelength. Incertain examples the signal is the disappearance of a physical event,such as quenching of light.

sNRF (soluble natriuretic peptide receptor-related fragment): Anintracellular fragment of NPRA that interferes with binding ofcGMP-dependent kinase I (PKG) to NPR. In some examples, sNRF does notinclude a kinase homology domain. In some examples, sNRF does notinclude a hinge domain.

In particular examples, a sNRF protein includes a sequence of at least60 contiguous amino acids from the intracellular region of NPRA, forexample at least 70 contiguous amino acids, at least 80 contiguous aminoacids, or at least 90 contiguous amino acids from the intracellularregion of NPRA (such as at least 60, at least 70, at least 80, or atleast 90 contiguous amino acids of residues 806 to 1061 of SEQ ID NO: 2or residues 820-1061 of SEQ ID NO: 2). Particular examples include, butare not limited to, the sequences provided in SEQ ID NOS: 4, 6, 38, 40and 42, as well as variants thereof that retain the biological activityof these fragments.

Subject: Living multicellular vertebrate organisms. Includes human andveterinary subjects, such as dogs, cats, cows, horses, sheep, rodents,and birds.

Test agent: Any peptide, organic compound, inorganic compound, nucleicacid molecule (such as an RNAi) or other molecule of interest. Inparticular examples, a test agent can permeate a cell membrane (alone orin the presence of a carrier). In particular examples, a test agent isone whose ability to treat one or more symptoms of heart failure isdesired to be determined, for example its ability to decrease thebiological activity of sNRF.

Therapeutically effective amount: An amount of an agent (alone or incombination with other therapeutically effective agents) sufficient toachieve a desired biological effect. In one example, it is an amountthat is effective to increase the activity of a NPRA/B, such as adesensitized NRPA receptor. In one example, it is an amount that iseffective to decrease the deleterious growth factor effects. Inparticular examples, increasing the activity of NPRA/B or decreasingdeleterious growth factor effects alters the production of anintracellular marker of NPRA/B function (such as cGMP or α-SMA),increases the sensitivity of NPR for NP ligands, such as in a CF cell ofa subject to whom it is administered. In a particular example, theactivity of NPRA/B is increased or the deleterious growth factor effectsdecreased (or both) by decreasing the activity of sNRF, for example bydecreasing expression of sNRF.

In a particular example, it is an amount of an agent effective toincrease the activity of NPRA/B by at least 25%, at least 50%, at least75%, or at least 90%, for example as compared to an amount of activityprior to treatment. In other or additional examples, it is an amounteffective to increase production of an intracellular marker of NPRA/Bfunction, such as increase in production of α-SMA by at least 25%, atleast 50%, at least 75%, or even at least 90% as compared to an amountof production prior to treatment. In other or additional examples, it isan amount effective to decrease production of an intracellular marker ofNPRA/B function, such as a decrease in production of cGMP by at least25%, at least 50%, at least 75%, or even at least 90% as compared to anamount of production prior to treatment. In other or additionalexamples, it is an amount effective to increase the sensitivity ofNPRA/B for NP ligands by at least 25%, at least 50%, at least 75%, or atleast 90% as compared to an amount of sensitivity of NPRA/B for NPligands prior to treatment. In some examples, it is an amount of anagent effective to decrease deleterious growth factor effects by atleast 25%, at least 50%, at least 75%, or at least 90%, for example ascompared to an amount of activity prior to treatment. In other oradditional examples, it is an amount effective to decrease thebiological activity of sNRF, such as the detectable expression of sNRF,by at least 25%, at least 50%, at least 75%, or at least 90% as comparedto an amount of activity prior to treatment.

In some examples, it is an amount of a therapeutic agent (alone or incombination with other therapeutically effective agents) that canincrease the activity of NPRA/B, decrease deleterious growth factoreffects, decrease the activity of sNRF, or combinations thereof, toimprove signs or symptoms of a disease caused by decreased NPRA/Bactivity. In particular examples a therapeutically effective amountimproves one or more signs or symptoms of cardiovascular disease, forexample such a condition associated with desensitized NPRA/B orincreased growth factor activity, such as heart failure.

An effective amount of an agent that increases the activity of NPRA/B,decrease deleterious growth factor effects, decrease the activity ofsNRF, or combinations thereof, can be administered in a single dose, orin several doses (for example daily, weekly, or monthly) during a courseof treatment. However, the effective amount of agent may be dependent onthe source of agent administered, the subject being treated, theseverity and type of disease being treated, and the manner ofadministration. For example, a therapeutically effective amount of atherapeutic agent disclosed herein (such as one that increases theactivity of NPRA/B or decreases deleterious growth factor effects) canvary from about 1 μg/kg body weight to about 20 μg/kg body weight perdose, about 1 μg/kg body weight to about 10 μg/kg body weight per dose,about 10 μg/kg body weight to about 20 μg/kg body weight per dose, orabout 1-2 μg agent/kg body weight/dose. In another example, atherapeutically effective amount of an RNAi nucleic acid molecule (suchas an siRNA, antisense, or miR) that decreases the activity of sNRF canvary from about 1 mg/kg body weight to about 100 mg/kg body weight perdose, about 1 mg/kg body weight to about 10 mg/kg body weight per dose,about 10 mg/kg body weight to about 20 mg/kg body weight per dose, orabout 1-2 mg/kg body weight/dose.

To assess restoration or increased NPRA/B activity, decreaseddeleterious growth factor effects, or combinations thereof, (for exampleto assess decreased sNRF biological activity), the methods disclosedherein can be used to compare a subject before and after treatment. Forexample, expression of intracellular markers of NPRA/B function,sensitivity of NPRA/B to NP ligands, and the effect on signs andsymptoms of cardiovascular disease can be determined using the methodsdescribed below. Similarly, the methods disclosed herein can be used tocompare a subject before and after treatment.

Transduce, Transform, or Transfect: To introduce a nucleic acid moleculeinto a cell, such as an siRNA or other inhibitor nucleic moleculespecific for sNRF. These terms encompass all techniques by which anucleic acid molecule can be introduced into a cell, including but notlimited to, transfection with viral vectors, transformation with plasmidvectors, and introduction of naked DNA by electroporation, lipofection,and particle gun acceleration. A transfected or transformed cell is acell into which has been introduced a nucleic acid molecule by molecularbiology techniques, such as a transformed CF that includes a recombinantpromoter operably linked to a reporter nucleic acid molecule. Inparticular examples the nucleic acid molecule becomes stably replicatedby the cell, for example by incorporation of the nucleic acid moleculeinto the cellular genome, or by episomal replication.

Treating a disease: “Treatment” refers to a therapeutic interventionthat ameliorates a sign or symptom of a disease or pathologicalcondition, such a sign or symptom of cardiovascular disease. Treatmentcan also induce remission or cure of a condition, such as acardiovascular disease. In particular examples, treatment includespreventing a disease, for example by inhibiting the full development ofa disease, such as preventing development of cardiovascular disease (forexample heart failure). Prevention of a disease does not require a totalabsence of cardiovascular disease. For example, a decrease of at least10%, at least 25% or at least 50% can be sufficient.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity.

In one example, includes culturing cells (such as CFs) sufficient toallow the desired activity. In particular examples, the desired activityis increasing the biological activity of NPRA/B in the cell, decreasingthe deleterious growth factor action on cardiovascular disease, orcombinations thereof.

In another example, includes administering an agent (such as oneidentified using the disclosed methods) to a subject sufficient to allowthe desired activity. In particular examples, the desired activity isincreasing the biological activity of NPRA in a cell (such as a CF),decreasing the deleterious growth factor action in a cell (such as aCF), or combinations thereof.

Unit dose: A physically discrete unit containing a predeterminedquantity of an active material (such as a therapeutic agent identifiedusing the disclosed methods) calculated to individually or collectivelyproduce a desired effect. A single unit dose or a plurality of unitdoses can be used to provide the desired effect, such as treatment ofheart failure.

Variant sequence: A native sequence, such as a native sNRF sequence,that is modified at one or more nucleotides or one or more amino acids.Exemplary variants include mutants (such as sequences that include oneor more nucleotide or amino acid substitutions, deletions, insertions,or combinations thereof), fragments (such as a fragment that retains thebiological activity of the native protein), fusions (for example fusionto a sequence that permits purification of a protein, such as aHis-tag), or combinations thereof. Ideally, variant sequences retain thebiological activity of the native sequence (for example the same abilityto disrupt PKG-NPRA association as the native sequence).

Vector: A nucleic acid molecule as introduced into a cell, therebyproducing a transformed cell. A vector can include nucleic acidsequences that permit it to replicate in the cell, such as an origin ofreplication. A vector can also include one or more selectable markergenes and other genetic elements known in the art. In a particularexample, a vector includes a promoter operably linked upstream from areporter nucleic acid sequence.

The pleiotrophic beneficial effects of natriuretic peptides (NPs),including diuresis, vasodilation, promotion of cardiomyocyte survival,inhibition of cardiac hypertrophy, inhibition of cardiac fibroblastproliferation, inhibition of smooth muscle proliferation, inhibition ofthe sympathetic nervous system, and inhibition of aldosterone synthesis,has fostered enthusiasm for the use of synthetic human NPs as a heartfailure therapy. However, in heart failure, the beneficial effects ofNPs, including inhibition of growth factor-induced cardiac fibrosis, areblunted. Heart failure itself was initially postulated to be anNP-deficient state. Later, when massively high circulating NP levelswere observed in heart failure patients, it became evident that theheart, vasculature, and kidneys were, in fact, NP-resistant. Themechanism of NP resistance in heart failure is currently unknown.

It is shown herein that a soluble, C-terminal fragment of the NPRA gene(named soluble natriuretic peptide receptor-related fragment, or sNRF)likely causes NP resistance. The sNRF mRNA is the result oftranscription initiation in exon 15 of the NPRA gene on human chromosome1, and encodes a cytosolic protein comprised of more than half of theintracellular portion of NPRA.

It is shown herein that cGMP-dependent protein kinase I (PKG) associateswith NPRA and phosphorylates it in a ligand-dependent fashion. Theassociation between PKG and NPRA is involved in the ligand-dependentreceptor guanylyl cyclase activation, as well as NPRA's ability toreduce or inhibit growth factor-induced CF differentiation. Theinteraction of PKG and NPRA can be disrupted in the presence of sNRF,indicating that the PKG-NPRA association is a component of NPRAactivation and that the failure or reduction of PKG-NPRA association isinvolved in the desensitization of NPRA in a variety of cardiacdiseases. When the interaction between PKG and NPR is disrupted, NPRbecomes insensitive to the presence of NP ligand, thus decreasing theactivity of NPR.

sNRF regulates NPRA activation and inhibits NPs' ability to reverse theharmful cardiac effects of FGF and transforming growth factor-β₁(TGF-β₁). sNRF appears to amplify TGF-β₁ effects that, in turn, maypromote heart failure. Thus, sNRF expression mimics the hormonal milieuof heart failure produced by NPRA knockout, and it is proposed that sNRFplays a role in the progression of heart failure. The observationspresented herein indicate that dysregulation of PKG-NPRA association andthe resulting inhibition of NPRA phosphorylation may be the mechanism ofclinical NPRA resistance. Without wishing to be bound to a particulartheory, it is proposed that in the presence of sNRF (which interfereswith the PKG-NPRA association), PKG can no longer interact with orphosphorylate NPRA. As a result, the presence of NP has no effect on theNPR. Alternatively sNRF may act by potentiating the deleterious effectof a growth factor, for example a TGFβ1 effect on cardiovasculardiseases, independently of sNRF effects on NPR.

Based on these observations, methods of treating heart failure byadministration of agents that interfere with sNRF biological activity(such as RNAi molecules specific for sNRF), agents that increase thebiological activity of NPR (such as NPRA), agents that inhibit thepotentiation of growth factor deleterious effects, or combinationsthereof. Also provided are methods of screening for agents that increasethe biological activity of NPR, agents that inhibit the potentiation ofgrowth factor deleterious effects, or both, such as sNRF-inhibitingsmall molecules. In particular examples, such agents can treat heartfailure by restoring or enhancing the beneficial effects of the NPsystem or inhibiting the deleterious effects of growth factors. Suchagents may increase the affinity of PKG for NPRA or NPRB (referred toherein as NPRA/B). In particular examples, these agents may activateNPRA/B independently of ligand, re-sensitize the receptor to ligandaction, decrease the biological effects of sNRF, decrease thedeleterious effects of growth factors, or combinations thereof. It isalso possible to treat heart failure by inhibiting interference of theNPRA interference with PKG-NPRA association, for example byadministering an inhibitor of sNRF. Similarly, it is possible to providemodels of heart failure in an animal by administering agents (such assNRF) that interfere with PKG-NPRA association, agents that increaseharmful growth factor actions, or combinations thereof.

Methods of Treating Cardiovascular Disease

Based on the results herein, it is proposed that prolonged NP exposureincreases sNRF expression, and that decreasing the biological activityof sNRF (or increasing the biological activity of NPRA or NPRB, forexample by increasing the sensitivity of NPRA for ANP) will rescue NPresponsiveness in human CFs. The present disclosure provides methods oftreating cardiovascular disease, for example by restoring NPs'beneficial effects in NP-resistant cells. In some examples, thecardiovascular disease to be treated results from a ligand-induceddesensitization of NPRA, results from other ill-effects of increasedsNRF expression (such as the harmful effects of growth factor actionthat are potentiated by sNRF expression), or combinations thereof. Inparticular examples, the method includes inhibiting or decreasing thebiological activity of sNRF (such as its ability to desensitize NPRA),increasing the biological activity of NPR (such as its sensitivity toNP), decreasing or inhibiting growth factor effects that are increasedby sNRF, or combinations thereof.

Particular cardiovascular diseases that can be treated using thedisclosed methods include, but are not limited to: angina pectoris;arrhythmia; cardiac fibrosis, congenital cardiac malformations; coronaryartery disease (CAD); dilated cardiomyopathy; heart attack (myocardialinfarction); heart failure; hypertrophic cardiomyopathy; systemichypertension from any cause, edematous disorders caused by liver orrenal disease, mitral regurgitation, myocardial tumors, myocarditis,rheumatic fever, Kawasaki disease, Takaysu arteritis, cor pulmonale,primary pulmonary hypertension, amyloidosis, hemachromatosis, toxiceffects on the heart due to poisoning, Chaga's disease, hearttransplantation, cardiac rejection after heart transplantation,cardiomyopathy of chachexia, arrhythmogenic right ventricular dysplasia,cardiomyopathy of pregnancy, or cardiovascular manifestations of MarfanSyndrome; Turner Syndrome; Loeys-Dietz Syndrome, familial biscuspidaortic valve, or any inherited disorder of the heart or vasculature, orcombinations thereof.

Inhibiting sNRF

Methods of inhibiting the biological activity of a nucleic acid orprotein sequence are known in the art. Although particular examples ofsuch methods are provided herein for illustrative purposes, thedisclosure is not limited to such methods. In particular examples,inhibiting the biological activity of sNRF does not require a 100%reduction. For example, decreases of at least 20%, at least 40%, atleast 50%, at least 60%, at least 75%, at least 80%, at least 90%, atleast 95%, or at least 99%, as compared to a control (such as an amountof activity in a cell not treated with a therapeutic agent), can besufficient.

One particular method that can be used to decrease the biologicalactivity of a sNRF nucleic acid or protein sequence is to decrease ordisrupt transcription or translation of an mRNA encoding sNRF in thecell. Decreased expression of sNRF will result in a decreased amount ofsNRF available for desensitizing NPR.

Based on the disclosed sNRF nucleic acid sequences (for example see SEQID NOS: 3, 5, 7, 37, 39, and 41), including variants, fusions andfragments of such sequences, methods that can be used to interrupt oralter transcription of such nucleic acid molecules include, but are notlimited to, site-directed mutagenesis (including mutations caused by atransposon or an insertional vector), providing a DNA-binding proteinthat binds to the coding region of the protein (thus blocking orinterfering with RNA polymerase or another protein involved intranscription), disrupting expression of sNRF coding sequence (forexample by functionally deleting the coding sequence, such as by amutation, insertion, or deletion), altering the amino acid sequence oroverall shape of sNRF protein, degrading sNRF protein, or combinationsthereof.

Various inactive and recombinant DNA-binding proteins, and their effectson transcription, are discussed in Lewin, Genes VII. Methods that can beused to interrupt or alter translation of a nucleic acid moleculeinclude, but are not limited to, using an antisense RNA, ribozyme or ansiRNA that binds to a messenger RNA transcribed by the nucleic acidencoding sNRF. Such methods can be used to decrease or inhibitexpression of sNRF, to treat heart failure.

For example, the amount mRNA can be decreased in the cell by contactingthe mRNA with a therapeutically effective amount of a molecule thatbinds to sNRF messenger RNA, for example molecules that arecomplementary to intron 15 of NPRA (nucleotides 1-215 of SEQ ID NO: 37).Examples of such complementary molecules include antisense RNA,ribozyme, triple helix molecule, miR, or siRNA that is specific for themRNA, for example by administering to the cell the antisense RNA,ribozyme, triple helix molecule, miR, or siRNA. In one example,antisense RNA, triple helix molecule, ribozyme, miR, or siRNA moleculesare contacted with the cell under conditions that permit the molecule tobe introduced into the cell. In a particular example, an expressionvector that transcribes one or more antisense RNA, ribozyme, triplehelix molecule, miR, or siRNA sequences that recognize a sNRF mRNAsequence is used to transform cells.

Particular siRNA, antisense, and ribozyme molecules are disclosedherein. For example, any of the disclosed siRNA molecules (14, 16, 18,20, 22, 24, 26, 28, 30, or 32), or combinations thereof, such as atleast 2, at least 3, or at least 4 of these sequences (such as 2, 3, 4,5, 6, 7, 8, 9 or 10 of these), can be used at therapeutically effectiveamounts to decrease an amount of sNRF mRNA in the cell. In one example,the therapeutic molecule is a duplex (such as a duplex s formed by SEQID NOS: 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and24, 25 and 26, 27 and 28, 29 and 30, or 31 and 32). However, thisdisclosure is not limited to the use of particular molecules thatdecrease mRNA.

Molecules that bind sNRF and prevent it from binding PKG or prevent itspotentiating effect on growth factor action action can be used to treata cardiovascular disorder, such as heart failure. In particularexamples, decreasing the biological activity of sNRF includes decreasingthe interaction between sNRF and PKG. For example, to decrease theinteraction between sNRF and PKG an agent that decreases, inhibits, ordisrupts the interaction (for example, a binding interaction) between asNRF and PKG can be administered (for example to a subject in need oftherapy). Agents that recognize sNRF or PKG or portion thereof, canprevent binding of sNRF and PKG, thereby decreasing or inhibiting NPRdesensitization or decrease sNRF's harmful effect on growth factoraction. Examples of such agents include, but are not limited to, ananti-protein binding agent that specifically binds to sNRF or PKG, suchas an antibody, peptide, or chemical.

Using Agents Identified by the Disclosed Screening Method

Agents identified using the methods disclosed herein can be used totreat a cardiovascular disorder. For example, one or more identifiedagents can be administered in a therapeutically effective amount to asubject having cardiovascular disease, or to a subject having anincreased risk for developing cardiovascular disease. Such agents can beadministered alone, or in the presence of a pharmaceutically acceptablecarrier. In addition, agents identified using the disclosed methods canbe administered in combination with other therapies.

Pre-Screening Subjects

In particular examples, the method includes determining whether thesubject has cardiovascular disease or is at an increased risk fordeveloping cardiovascular disease. Methods of determining whether asubject has heart disease or has an increased risk of developing heartdisease in the future are known in the art. For example, serum levels ofANP, BNP, or cGMP can be detected in a sample obtained from the subject,wherein a serum level of >50-100 pg/ml for ANP, a serum level of >50-10pg/ml for BNP, or serum level >8 pg/ml for cGMP, indicates that thesubject has cardiovascular disease or is at an increased risk fordeveloping cardiovascular disease.

Subjects known or found to have cardiovascular disease or an increasedrisk for developing cardiovascular disease are then administered atherapeutically effective amount of the therapeutic agent(s) disclosedherein.

Modes of Administration and Dosages

In one example, a therapeutically effective amount of an agent thatdecreases the biological activity of sNRF (for example by decreasingexpression of sNRF mRNA) or increases the biological activity of NPRA/B,or decreases deleterious growth factor affects, or combinations thereof,is contacted with a cell, for example by administration to a subject.Such an agent can be used for prophylactic or therapeutic purposes.

For example, antisense oligonucleotides, ribozymes, triple helixmolecules, miRs, and siRNA molecules that recognize sNRF can beadministered to the subject to disrupt expression of sNRF. In aparticular example, an expression vector including antisense RNA,ribozyme, triple helix molecule, miR, or siRNA molecules that targetssNRF nucleic acid sequence is introduced intravenously to a subject in atherapeutically effective amount. Uptake of the vector and expression ofthe antisense RNA, ribozyme, triple helix molecule, miR, or siRNA withincardiac cells (such as CF cells), offers a prophylactic or therapeuticeffect by decreasing expression of sNRF within those cells, thustreating the cardiovascular disorder. In particular examples, expressionof the antisense RNA, ribozyme, triple helix molecule, miR, or siRNA isunder control of a promoter, such as an inducible promoter. The vector,or other nucleic acid molecule, can be introduced into a subject by anystandard molecular biology method and can be included in a compositionthat includes a pharmaceutically acceptable carrier.

Screening Assays

Natriuretic peptides (NPs) are hormones produced by the heart thatcounteract heart failure pathways, such as total body fluid overload,over-activation of several hormones (such as angiotensin, aldosterone,endothelin, renin, the sympathetic nervous system, programmed cell death(apoptosis) and activation of inflammatory cytokines), and maladaptivethickening of the heart muscle (hypertrophy). However, ultimately, NPaction is overwhelmed, chronic illness or and death can ensue. In thissituation, called decompensated heart failure, despite very highcirculating NP levels, NP actions are blunted, in part because the NPR(such as NPRA/B) becomes resistant to circulating NPs. Therefore,treatments are needed that overcome this ligand-induced NPRdesensitization. For example, agents that permit reinvigoration of theNPRA/B, such that it becomes either responsive to the high levels of NPsin the circulation (for example by increasing the sensitivity of thereceptor for NP ligands such as ANP or BNP), could be used to treatcardiovascular disease, such as heart failure.

The results provided herein demonstrate that sNRF controls NPRAresistance to the presence of NPs and potentiates the harmful effects ofcertain growth factors (such as FGF and TGFβ1). Therefore, sNRF providesa target for the identification of therapeutic agents. Specifically,sNRF and variants thereof have been identified that are capable ofmodulating NPRA's sensitivity to NPs or modulating the harmful action ofcertain growth factors (such as FGF and TGFβ1). These peptides associate(either directly or indirectly via other molecules) with PKG. It isshown herein that NPRA associates with PKG, and that association isinvolved in for maintaining the structure and function of the normalreceptor complex. Thus, sNRF behaves as a decoy peptide or competitiveinhibitor of the interaction between PKG and NPRA that maintainsresponsiveness of the receptor to NP ligands or behaves as acarrier/binding protein that regulates the sub-cellular location of PKGin the cell. It is also shown herein that sNRF affects downstream NPaction. For example, sNRF represses NP's inhibitory action on cardiacfibroblast proliferation and differentiation through its ability tocompetitively bind PKG.

Based on these observations, methods are provided for screening testagents for their ability to increase the biological activity of anatriuretic peptide receptor (NPR), such as NPRA/B, to decreasesNRF-induced potentiation of harmful growth factor effects, orcombinations thereof. Although monogenetic mouse models of NPRAdysfunction can be used to identify agents that treat heart failure,unless they are engineered to conditionally regulate the gene ofinterest, the analysis of down stream effects is complicated byredundant systems that continue to be expressed as the animal develops.The reductionist approach taken here permits a focused analysis of NPRAdesensitization.

Exemplary test agents include proteins, nucleic acid molecules (such asRNAi) organic compounds, or inorganic compounds. In one example,increasing the biological activity of NPR, such as NPRA/B, includesincreasing the sensitivity of the receptor for NPs (such as ANP or BNP).For example, an increase in the sensitivity of NPRA/B for ANP or BNP inthe presence of the test agent, such as an increase of at least 25%, atleast 50%, at least 90%, or at least 95%, indicates that the test agentcan increase the biological activity of NPRA/B. In particular examples,this increase is relative to a reference value or a control, such as anamount of activity in the absence of the test agent. Methods formeasuring the sensitivity of NPR for NP ligands are disclosed herein.For example a cGMP assay can be used.

In particular examples, the method includes contacting one or more testagents, growth factors, and NPs, with a cell under conditions thatpermit the test agent to interact with NPRA/B present in the cell.Exemplary cells include cardiac cells, such as cardiac myocytes orcardiac fibroblasts (CF). Subsequently, a determination as to whetherthe test agent increased biological activity of the NPRA/B, or decreasedsNRF-induced potentiation of harmful growth factor effects, or both, ismade. The cells include a reporter (such as a recombinant nucleic acidmolecule that encodes a protein that permits for a determination ofNPRA/B biological activity), NPRA or NPRB, and an intracellular fragmentof NPRA that interferes with binding of cGMP-dependent kinase I (PKG) toNPR. The NPRA or NPRB in the cell can be native or recombinant.

In a particular example, the reporter is a recombinant promoter operablylinked to a reporter nucleic acid sequence. For example, the promotercan be responsive to growth factors that are modulated by NP. In anotherexample, the reporter is a recombinant CNG channel expressed in thecell, whose activity can be detected by measuring the presence of Ca²⁺or Mn²⁺, for example using fura-2.

Many different assays are available for determining whether the testagent increased biological activity of NPRA/B, decreased sNRF-inducedpotentiation of harmful growth factor effects, or combinations thereof.In one example, the method includes detecting a signal generated from areporter (such as protein encoded by a reporter nucleic acid sequence orthe influx of Ca²⁺ or Mn²⁺ through a recombinant CNG channel), wherein achange in the signal compared to the signal present in an absence of thetest agent indicates that the test agent is an agent that increasesbiological activity of the NPRA/B, decreases sNRF-induced potentiationof harmful growth factor effects, or combinations thereof. For example,the presence of a decreased signal in the presence of the test agent ascompared to a reference signal (such as a signal present in the absenceof the test agent) indicates that the test agent is an agent thatincreases biological activity of NPRA/B. As the mechanism ofsNRF-induced potentiation of harmful growth factor effects is not likelycGMP, inhibition of this pathway would not likely result in a decreasein cGMP. Therefore, while the use of a luciferase reporter (or otherreporter operably linked to α-SMA) would show “quenching” of the signalin the presence of a sNRF-inhibiting agent (such as one that decreasedsNRF-induced potentiation of harmful growth factor effects), the use ofa CNG channel (that depends on modulation of cGMP) would not beaffected.

In a particular example, the signal is a colorimetric signal (such asfluorescence or luminescence), or expression of a growth supplement inthe presence of a nutrient deficient culture medium. Methods ofdetecting such signals are known in the art, and can include, but arenot limited to, ELISA, spectrophotometry, flow cytometry, or microscopy.

In one particular example, the assay is a method for identifying agentsthat increase the biological activity of NPRA, NPRB, or both. In someexamples, the method includes contacting a cell (such as a CF cell) withone or more test agents, with TGF-β, and with ANP or BNP. In specificexamples, the cells include a recombinant α-SMA promoter operably linkedupstream to a reporter nucleic acid sequence, NPRA/B, and a recombinantsequence comprising or consisting of SEQ ID NO: 4, 6, 38, 40 or 42. Themethod further includes measuring a signal produced by a protein encodedby the reporter nucleic acid sequence (such as luciferase) anddetermining whether the signal produced in the presence of the testagent is altered as compared to the signal in the absence of the testagent, where a change in the signal indicates that the test agentincreases biological activity of NPRA, NPRB, or both. Such agents can beselected for further analysis, and in some examples are used to treatcardiovascular disease.

In another particular example, the assay is a method for identifyingagents that increase the biological activity of NPRA, NPRB, or both. Insome examples, the method includes contacting a cell (such as a CF cell)with one or more test agents, with TGF-β, and with ANP or BNP. Inspecific examples, the cells include a recombinant CNG channel (forexample a cell infected with an adenovirus encoding the CNGA2 subunit,see Fagan et al., J. Biol. Chem. 274:12445-53, 1999, herein incorporatedby reference), NPRA/B, and a recombinant sequence comprising orconsisting of SEQ ID NO: 4, 6, 38, 40 or 42. The method further includesmeasuring a signal produced by influx of Ca²⁺ or Mn²⁺ (for example bymeasuring intracellular Ca²⁺ or Mn²⁺ using fura 2) and determiningwhether the signal produced in the presence of the test agent is alteredas compared to the signal in the absence of the test agent, where achange in the signal (such as an increase or decrease) indicates thatthe test agent increases biological activity of NPRA, NPRB, or both.Such agents can be selected for further analysis, and in some examplesare used to treat cardiovascular disease.

In another particular example, the assay is a method for identifyingagents that decrease sNRF-induced potentiation of harmful growth factoreffects. In some examples, the method includes contacting a cell (suchas a CF cell) with one or more test agents, with TGF-β, and with ANP orBNP. In specific examples, the cells include a recombinant α-SMApromoter operably linked upstream to a reporter nucleic acid sequence,NPRA/B, and a recombinant sequence comprising or consisting of SEQ IDNO: 4, 6, 38, 40 or 42. The method further includes measuring a signalproduced by a protein encoded by the reporter nucleic acid sequence(such as luciferase) and determining whether the signal produced in thepresence of the test agent is altered as compared to the signal in theabsence of the test agent, where a change in the signal indicates thatthe test agent decrease sNRF-induced potentiation of harmful growthfactor effects. Such agents can be selected for further analysis, and insome examples are used to treat cardiovascular disease.

In another particular example, the assay is a method for identifyingagents that decrease sNRF-induced potentiation of harmful growth factoreffects. In some examples, the method includes contacting a cell (suchas a CF cell) with one or more test agents, with TGF-β, and with ANP orBNP. In specific examples, the cells include a recombinant CNG channel(for example a cell infected with an adenovirus encoding the CNGA2subunit, see Fagan et al., J. Biol. Chem. 274:12445-53, 1999, hereinincorporated by reference), NPRA/B, and a recombinant sequencecomprising or consisting of SEQ ID NO: 4, 6, 38, 40 or 42. The methodfurther includes measuring a signal produced by influx of Ca²⁺ or Mn²⁺(for example by measuring intracellular Ca²⁺ or Mn²⁺ using fura 2) anddetermining whether the signal produced in the presence of the testagent is altered as compared to the signal in the absence of the testagent, where no significant change in the signal (such as an increase ordecrease) indicates that the test agent decreases sNRF-inducedpotentiation of harmful growth factor effects.

In another particular example, the method includes contacting a cell(such as a CF cell) with one or more test agents, with ANP or BNP, andoptionally a growth factor.

In some examples, the disclosed assays are performed in a multiple wellplate, such as a 6-, 12-, 24-, 96-, 384-, or 1536-well plate. In suchexamples, the cells are grown in the wells of the plate using standardtissue culture methods, and the desired agents (such as the test agents,growth factors, and NPs) added to the wells.

In some examples, test agents observed to increase the biologicalactivity of NPRA/B, decrease sNRF-induced potentiation of harmful growthfactor effects, or both, are selected. For example, such agents can besubjected to further analysis. In some examples such agents can be usedto treat cardiovascular disease.

In addition to the disclosed in vitro assays, the method can includefurther analysis of the test agents found in vitro to increase thebiological activity of the NPRA/B, decrease sNRF-induced potentiation ofharmful growth factor effects, or both. For example, test agents thatincrease the biological activity of the NPRA/B, decrease sNRF-inducedpotentiation of harmful growth factor effects, or both, can beadministered to a laboratory mammal having cardiovascular disease, anddetermining whether the test agent treats the cardiovascular disease.Animal models of cardiovascular disease are known in the art. Any routeof administration can be used. Particular doses and routes can bedetermined by those skilled in the art. In some examples, a wide rangeof concentrations of the test agents are used (such as 1 nM-1 mM) fortoxicity and LD₅₀ determinations.

Also provided by the present disclosure are agents identified using thedisclosed methods.

Cells

Any type of cell can be used in the disclosed assay, as long as the cellexpresses NPRA, NPRB (or both NPRA), as well as a sNRF that interfereswith binding of (PKG) to NPRA. In particular examples, the cellexpresses native NPRA, NPRB (or both), such as a cardiac cell. Examplesof cardiac cells that can be used include cardiac myocytes or CFs.However, even in a cardiac cell, levels of NPRA/B can be increased byrecombinantly expressing NPRA/B. In non-cardiac cells, such as HEK orCos 7 cells, the NPRA/B can be expressed recombinantly.

CFs express morphological and functional features of smooth muscle cellswhen the heart is stressed. These differentiated cells, calledmyofibroblasts, express microfilaments that are the force-generatingelements in wound contraction (Hinz et al., Mol Biol Cell. 14:2508-19,2003) and play a role in the cardiac response to myocardial infarction(Willems et al., Am. J. Pathol. 145:868-75, 1994), heart failure (Jaffeet al., Adv. Exper. Med. Biol. 430:257-66, 1997), and pulmonary veinstenosis in patients with cardiac malformations (Sadr et al., Am. J.Cardiol. 86:577-9, A10, 2000). The cytoskeletal protein alpha smoothmuscle actin (α-SMA) has a mechanistic role in this process, and is alsothe principal molecular marker of CF differentiation intomyofibroblasts. Undifferentiated CFs express NPRA but minimal NPs. Whenanimals are stressed by experimental myocardial infarction or when CFsare grown in tissue culture and treated with growth factors such asTGF-β or FGF, CFs transdifferentiate into myofibroblasts, begin toexpress NPs, and express significant amounts of α-SMA. Thus, primarycultures of CFs are a model for screening test agents for their abilityto increase biological activity of NPRA/B.

In particular examples, the CF used is a primary CF cell isolated from amammal. For example, laboratory mammals (such as rats, mice, andrabbits) can be used as a source of cardiac tissue from which primaryCFs can be obtained. Methods of generating CF cells from tissue areknown in the art, and particular examples are provided herein.

One skilled in the art will appreciate that CF cells can be obtainedfrom other sources, such as a tissue culture cell line. In one example,a CF cell expresses endogenous NPRA or NPRB. In other examples, a CFcell expresses recombinant NPRA or NPRB.

Promoters

In examples where the reporter includes a promoter operably linked to areporter nucleic acid sequence, the promoter is ideally responsive tothe presence of one or more growth factors that are modulated by NP. Forexample, the promoter can be one that is activated in the presence of agrowth factor, wherein the activity of the growth factor is reduced inthe presence of NP (such as ANP).

Particular examples of promoters that can be used include, but are notlimited to: alpha smooth muscle actin (α-SMA) promoter, pro alpha 2(I)collagen promotor, β-myosin heavy chain promotor, or the atrialnatriuretic peptide promotor itself.

Placement of the promoter upstream of the reporter can be achieved usingstandard molecular biology methods. In a particular example, thepromoter is operably linked to the reporter and the resulting constructis part of a vector, which is introduced into CF cells using standardtransformation methods.

Reporter Molecules

Reporter nucleic acid sequences operably linked to a promoter responsiveto the presence of one or more growth factors that are modulated by NPinclude are those that encode a protein that produces a detectablesignal when expressed, such as a colorimetric signal (for example aluminescent or fluorescent signal). Expression of a reporter can becontrolled by a promoter sequence upstream of

The reporter nucleic acid can include a promoter, the structuralsequence of the reporter gene, and the sequences required for theformation of functional mRNA. Upon introduction of the reporterconstruct into cells, expression levels of the reporter gene can bemonitored, for example by assaying for the reporter protein's enzymaticactivity, or by measuring production of the protein directly (forexample if the protein is a fluorescent or luminescent protein, such asgreen fluorescent protein (GFP), fluorescence or luminescence can bedetected).

Particular examples include, but are not limited to: luciferase,β-galactosidase, chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP) (or variants thereof such as E-GFP). Sequencesfor such reporter molecules are well-known in the art. For example, apromoter that is responsive to the presence of one or more growthfactors modulated by NP can be inserted into these plasmids that includethe indicated reporter: p-lacZ (beta-galactosidase reporter), p-luc(firefly luciferase reporter) and p-cat (chloramphenicol acetyltransferase).

CNG Channels

In one example, the cell used in the disclosed in vitro assays includesa recombinant CNG channel (or functional subunit thereof, such as CNGA2)as the reporter. In such examples, the activity of NPRA/B is detected bymeasuring the presence of intracellular calcium (for example an increasein fura 2 fluorescence indicates that the NPRA/B is biologicallyactive), or by measuring the quenching of fura 2 by manganese (forexample a decrease in fura 2 fluorescence indicates that the NPRA/B isbiologically active).

sNRF Molecules

sNRF peptides that interfere with binding of PKG to NPR or potentiatethe action of growth factors can be used to identify agents thatincrease the biological activity of NPR, reduce the harmful effects ofgrowth factors in cardiovascular disease, or combinations thereof. Theability of sNRF to interfere with binding of PKG to NPR or to potentiatethe action of growth factor can be determined using the methodsdisclosed herein. For example, immunoblotting and immunofluorescencemicroscopy (see Examples 7 and 10) can be used. However, it should benoted that it is not required that sNRF completely interfere withbinding of PKG to NPR. For example, a reduction in detectable PKG-NPRAcomplexes of at least 80% can be sufficient, such as a reduction of atleast 90% or at least 95%, or 100%, if sNRF acts by binding PKG andinterfering with other PKG actions (for example interfering with PKGinhibition of thrombospondin expression). Similarly, in some examplesthe ability of sNRF to potentiate the harmful actions of a growth factor(such as FGF or TGFβ1) on cardiovascular disease can be an increase inthe harmful actions of at least 10%, such as an increase of at least 20%or at least 50%.

The present disclosure provides sNRF sequences that interfere withbinding of PKG to NPRA, such as SEQ ID NO: 4, 6, 38, 40 or 42. Alsoprovided are nucleic acid molecules that encode such fragments. Inparticular examples, sNRF includes SEQ ID NO: 4, 6, 38, 40 or 42. Inother particular examples, sNRF consists of SEQ ID NO: 4, 6, 38, 40 or42. In some examples, sNRF includes at least 60 contiguous amino acidsof the NPRA intracellular domain, such as at least 60 contiguous aminoacids starting at amino acid 806 or 820 of SEQ ID NO: 2, for example atleast 70 contiguous amino acids starting at amino acid 806 or 820 of SEQID NO: 2, at least 80 contiguous amino acids starting at amino acid 806or 820 of SEQ ID NO: 2, or at least 90 contiguous amino acids startingat amino acid 806 or 820 of SEQ ID NO: 2. In particular examples, sNRFpeptides that interfere with binding of PKG to NPRA do not include anNPRA kinase-like domain. In other or additional examples, sNRF includesan NPRA hinge domain. The location of such domains in NPRA are known inthe art.

Although particular examples of sNRF that interfere with binding of PKGto NPR are disclosed, the assay is not limited to use of thesesequences. For example, variants of SEQ ID NO: 4, 6, 38, 40 or 42 can beused. Particular examples of variants include the amino acid sequenceshown in SEQ ID NO: 4, 6, 38, 40 or 42 having 1-10 conservative aminoacid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10conservative amino acid substitutions. Particular conservative aminoacid substitutions that can be made to SEQ ID NO: 4, 6, 38, 40 or 42include but are not limited to: R1060K, F1030Y, S892T, or combinationsthereof (wherein numbering refers to SEQ ID NO: 2). Particular aminoacid substitutions (not necessarily conservative) that can be made toSEQ ID NO: 4, 6, 38, 40 or 42, include but are not limited to themutations shown in FIG. 5 of Thompson and Garber (J. Biol. Chem.270:425-30, 1995). Other variants of SEQ ID NO: 4, 6, 38, 40 or 42include amino acid sequences that include 1-10 amino acid insertions ordeletions to SEQ ID NO: 4, 6, 38, 40 or 42.

Methods of introducing a sNRF that interferes with binding of PKG to NPRinto cells can be achieved using standard molecular biology methods. Forexample, a nucleotide sequence encoding for sNRF can be part of a vector(for example downstream from a promoter sequence), which can beintroduced into a cell, thereby allowing expression of sNRF in the cell.Particular examples of vectors and transfection methods are disclosed;however those skilled in the art can readily adapt these teachings touse other vectors or transfection methods.

Growth Factors

Growth factors that can be used in the disclosed methods include thosewhose biological activity (such as expression) are altered during heartfailure, and are modulated by NP. For example, growth factors includethose whose biological activity (such as expression) is increased duringheart failure, but whose biological activity is decrease in the presenceof NP, such as ANP. In particular examples sNRF expression potentiatesgrowth factor-induced production of α-SMA.

Particular examples of growth factors that can be used include, but arenot limited to: FGF, EGF or TGF-β.

Methods of Diagnosing Cardiovascular Disease

Also provided by the present disclosure are methods of diagnosingcardiovascular disease.

In one example, the disclosed sNRF molecules are used to determine if asubject has cardiovascular disease (such as heart failure), or evendetermine the severity of cardiovascular disease. The method can includedetermining whether the subject expresses one or more of the disclosedsNRF molecules. If the subject expresses one or more of the disclosedsNRF molecules above a baseline or reference level (such as a levelexpected in a subject not having cardiovascular disease), this indicatesthat the subject has cardiovascular disease or has an increased risk ofdeveloping such disease. In particular examples, if the subjectexpresses any of SEQ ID NOS: 4, 6, 8, 38, 40 or 42 (or combinationsthereof) above a baseline or reference level, this indicates that thesubject has cardiovascular disease or has an increased risk ofdeveloping such disease. In other particular examples, if the subjectexpresses more of SEQ ID NO: 4 than SEQ ID NO: 6 or SEQ ID NO: 8, moreof SEQ ID NO: 6 than SEQ ID NO: 8, more of SEQ ID NO: 40 than SEQ ID NO:42, more of SEQ ID NO: 4 than SEQ ID NO: 42, more of SEQ ID NO: 40 thanSEQ ID NO: 6, or more of SEQ ID NO: 40 than SEQ ID NO: 8, than thisindicates a more severe form of cardiovascular disease.

Methods of determining whether a subject expresses sNRF (such as any ofSEQ ID NOS: 4, 6, 8, 38, 40 or 42) are known in the art. For example,immunoassays and nucleic acid methods (such as Northern or Southernhybridization, PCR (for example quantitative PCR), and ELISA), can beused. In particular examples, a sample from the subject, such as a bloodsample (or fraction thereof), cardiac tissue sample (for example fromthe ventricle), saliva sample, urine sample, or cheek swab sample, isanalyzed for the presence of sNRF. If desired, the sample can beconcentrated or purified before use. In a particular example, the sampleincludes cardiac tissue.

In one example, subjects with latent or subclinical cardiovasculardisease will respond to a therapeutically effective amount of one ormore therapeutic agents identified using the disclosed methods. Thosenot responding would not have dysregulation of NPR-PKG association, donot have sufficient sNRF-induced potentiation of deleterious growthfactor effects, or combinations thereof. In a clinical setting an agentis said to increase the activity of NPRA/B (such as desensitized NPRA/B)when treatment of a subject with the agent results in the typicalresponse of a patient as if the NPRA/B is not desensitized. In anotherclinical setting, an agent is said to decrease the activity of a growthfactor (such as the deleterious effect of FGF or TGFβ1) when treatmentof a subject with the agent results in the typical response of a patientas if growth factor action is not increased. Such subjects might havenormalized blood pressure, increased urine output, suppression ofnumerous neuroendocrine markers of heart failure such as angiotensin,aldosterone, endothelin, renin, the sympathetic nervous system, or othergrowth factors, combing programmed cell death (apoptosis), decreasedcardiac fibrosis, decrease in cardiac filling pressures, improvement ofcardiac output, lessening of angina pectoris, normalization enlarged oraneurismal blood vessels, and in general diminution of the typical signsand symptoms of cardiovascular disease.

sNRF Proteins and Nucleic Acid Molecules

The present disclosure provides sNRF protein and nucleic acid sequences.For example, exemplary sNRF protein sequences are shown in SEQ ID NOS:4, 6, 38, 40 and 42, and exemplary sNRF nucleic acid sequences are shownin SEQ ID NOS: 3, 5, 37, 39 and 41. Therefore, the disclosure providesisolated proteins that include or consist of any of SEQ ID NOS: 4, 6,38, 40 or 42 as well as isolated nucleic acid sequences that include orconsist of any of SEQ ID NOS: 3, 5, 37, 39 and 41.

However, the disclosure is not limited to these exact sequences, as oneskilled in the art will appreciate that variants thereof can retain thesame biological activity as these sequences. For example, a sNRF proteinsequence can include one having at least 90%, at least 95%, or at least98% sequence identity to any of SEQ ID NOS: 4, 6, 38, 40 and 42, as longas the variant retains the biological activity of sNRF, such as theability to potentiate harmful growth factor effects, to disrupt PKG-NPRbinding, or combinations thereof. Similarly, variants of the nucleicacid molecules shown in SEQ ID NOS: 3, 5, 37, 39 and 41 can still encodethe identical amino acid sequence (for example due to the degeneracy ofthe code). In addition, variants of the disclosed sNRF nucleic acidmolecules can encode a different amino acid sequence, but still encode asNRF protein that retains sNRF biological activity. For example, a sNRFnucleic acid sequence can include one having at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% sequence identity to any ofSEQ ID NOS: 3, 5, 37, 39 and 41, as long as the variant encodes aprotein that retains the biological activity of sNRF, such as theability to potentiate harmful growth factor effects, to disrupt PKG-NPRbinding, or combinations thereof. Methods for testing sNRF biologicalactivity are disclosed herein.

Example 1 NPRA is Desensitized During Heart Failure

This example describes an in vitro model that mimics desensitization ofNPRA observed during heart failure.

Neonatal rat CFs were transfected with an α-SMA-luciferase reporterconstruct driven by TGF-β₁ induction of the α-SMA promoter. After 24hours, cells were treated daily with 100 nM TGF-β₁ for up to 4 days. Oneach successive day, some of the cells were treated with NP (ANP, 100nM), and luciferase activity measured.

As shown in FIG. 1, expression of the luciferase reporter construct,driven by TGF-β₁ induction of the α-SMA promoter, was almost completelyinhibited after 24 hours of NP treatment. However, progressively longerperiods of NP treatment resulted in a steady decline in NP's inhibitoryeffects, indicating that NPRA is desensitized.

It is proposed herein that sNRF activity accounts for this NPresistance.

Example 2 sNRF is a Fragment of NPRA

This example describes the sNRF fragment of NPRA.

The genomic sequence of NPRA comprises 22 exons and introns (FIG. 2). A816-bp partial NPRA cDNA sequence (SEQ ID NO: 37) contains the intron-15sequence (nucleotides 1-215 of SEQ ID NO: 37), followed by contiguousexon 16-20 sequences (nucleotides 216-816 of SEQ ID NO: 37, with thecoding sequence starting at nucleotide 256). This indicates that themRNA is a fully processed, alternative transcript of the NPRA gene. The215-nt, intron-15 sequence in this cDNA contains a stop codon 90 basesupstream of the 5′ end of NPRA exon 16 that is in-frame with the NPRAopen reading frame. The presence of this stop codon predicts that theprotein encoded by this novel transcript would initiate at an in-frameAUG codon 45 bases downstream of the 5′ end of exon 16. This NPRAfragment has been termed sNRF (soluble Natriuretic peptideReceptor-related Fragment).

Exemplary sNRF cDNA and protein sequence are provided in SEQ ID NOS: 39and 40 (sNRF⁽⁸²⁰⁻¹⁰⁶¹⁾), SEQ ID NOS: 41-42 (sNRF⁽⁸²⁰⁻⁹⁰⁰⁾), SEQ ID NOS:3 and 4 (sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾), and SEQ ID NOS: 5 and 6 (sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾).

Example 3 sNRF cDNA and RNA Analysis

This example describes methods used to confirm that sNRF is analternative NPRA gene-derived transcript initiating in intron 15, and ispresent in human cells.

RT-PCR of cDNA reversed-transcribed from total RNA extracted from humankidney and human genomic DNA was performed using primers correspondingto the intron-15 sequence and a 3′ segment of the exon-17 sequence (see“a” and “b” in FIG. 2, SEQ ID NOS: 43 and 44, respectively). Theresulting PCR product from human kidney cDNA had the predicted size of389 bp, whereas the PCR product of human genomic DNA was 477-bp,presumably containing the additional 88-nt, intron-17 sequence.

An intron 15-specific probe (SEQ ID NO: 35) was random-primed with³²P-ATP and used to probe a Northern blot containing total RNA fromnormal human heart and RNA from three heart explants prior to cardiactransplantation. The 18S ribosomal bands were of equal intensity whenthe two groups were compared (normal versus failing heart). However, asshown in FIG. 3, a strongly hybridizing band was observed in failingheart myocardium, but not in normal heart. This 1.5-kb band isconsistent with the size predicted for an alternative NPRA gene-derivedtranscript initiating in intron 15 (full-length NPRA mRNA is 4 kb).Therefore, sNRF and full-length NPRA transcript levels varyindependently in human heart.

These results demonstrate that sNRF is a naturally occurring transcriptpresent in the human heart.

Example 4 Differential sNRF Expression in Human Heart Failure

This example describes methods used to compare sNRF expression betweennormal hearts and heart failure hearts.

The increased expression of sNRF mRNA observed in the Northern blot ofpooled samples of failing myocardium (FIG. 3) indicates that sNRF isup-regulated in heart failure. To confirm this hypothesis, sNRF mRNAexpression was measured in five additional explanted hearts prior toheart transplantation using quantitative, real-time RT-PCR.

cDNA from reverse-transcribed poly(A)⁺ RNA from a transplant patientwith dilated cardiomyopathy (DCM) was compared to a control sampleobtained from another patient shortly after death from a non-cardiaccause. Quantitative, real-time PCR was performed using primers “a” and“b” shown in FIG. 2 (SEQ ID NOS: 43 and 44). Each sample was measured intriplicate using a Taqman probe (SEQ ID NO: 36, wherein the 5′ label isFAM and the 3′ label is TAMRA) with values adjusted relative to anendogenous GAPDH control using an ABI 7300 real-time PCR system. Asshown in FIG. 4A, a 5.7-fold increase in sNRF mRNA was observed in theDCM heart compared to the “normal” heart.

Total RNA was extracted from an explanted heart from a patient withhypertrophic cardiomyopathy. In that heart, sNRF mRNA expression wasnearly 70-fold higher when compared to RNA obtained from a “normal”heart (Ambion) (FIG. 4B, upper panel). The relative expression of thefull-length NPRA transcript was measured using a probe that spans exonicsequence upstream of sNRF (i.e., a probe complementary to exon 7 and 8exonic sequence not present in sNRF). There was essentially nodifference between full-length NPRA expression in the control heartcompared to the patient with HCM (FIG. 4B, lower panel).

In three additional explanted failing hearts, sNRF and NPRA transcriptsalso exhibited independent changes in expression. Relative sNRFexpression was 1, 4, and 21-fold, whereas NPRA expression levels were1.5, 1, and 1.3-fold, respectively, in each of the failing hearts (FIG.4C).

Total RNA extracted from 17 explanted pediatric hearts prior totransplantation was analyzed for sNRF expression as described above.Highly variable sNRF expression was also observed in myocardial samplesobtained from children with a variety of cardiomyopathies (FIG. 4D).

Taken together, these data indicate that regulation of sNRF expressionfrom its putative promoter in intron 15 is independent of NPRAexpression initiated at the NPRA promoter flanking exon 1.

Example 5 sNRF and NPRA Expression in Normal and Diseased Heart Tissue

This example describes methods used to compare sNRF and full-length NPRAexpression between normal hearts and diseased hearts. As shown in FIG.4B, differential expression of sNRF, but not NPRA, was detected innormal versus diseased heart tissue. To confirm this result, more heartswere analyzed.

sNRF and full-length NPRA mRNA levels in the left ventricles of patientswith and without heart failure were measured to demonstrate that sNRFgene expression correlates with heart failure. Using quantitative RT-PCRboth sNRF and full-length NPRA gene expression were compared in 7explanted hearts from patients with heart failure (patients receiving atransplant) to 4 normal hearts using the methods described in Example 4.

One skilled in the art will appreciate that “normal” heart tissue can beobtained from patients without heart failure as the primary diagnosis.For example, heart tissue can be obtained during surgery, for examplefrom coronary artery bypass grafting where small amounts of muscle areremoved when coronary arteries are dissected to connect the graft,septal myectomy for subaortic obstruction, ventricular septal defectenlargement during Rastelli-type procedures, and subaortic conal muscleresection in double-outlet right ventricle. In addition, commercial“normal” human heart mRNA is available (Ambion, Clontech, Biochain, andothers).

For each subject, serum brain-type natriuretic peptide (BNP) andC-reactive protein (CRP) (an inflammatory marker that correlates withheart failure severity) levels were determined, as well as clinicalheart failure NYHA status. Other data, including 6-minute walk, exerciseVo₂, echocardiographic parameters including left-ventricular mass, enddiastolic dimensions, fractional shortening and ejection fraction, canbe recorded. “Normal” patients presumably occupy NYHA classes I-3, while“heart failure” patients, having been listed for orthotopic hearttransplantation, were NYHA class IV.

At the time of surgery, myocardial samples were flash-frozen in liquidnitrogen within seconds of removal and stored at −80° C. Total mRNA wasextracted according to the manufacturer's protocol (RNeasy, Qiagen).Using this technique 10 μg of RNA per 10 mg of heart tissue wasobtained. Quantitative RT-PCR in triplicate using sNRF-specific primers(“a” and “b”, FIG. 2) and full-length NPRA-specific primers directed atexonic sequences upstream of the sNRF-unique region was performed asdescribed in Example 4. In rare cases where less than 10 mg is availablefrom a single patient, tissue from patients of equivalent NYHAclassification can be pooled. Quantitative RT-PCR was used to measureeither sNRF mRNA expression relative to GAPDH as an endogenous controlor absolute mRNA levels based on interpolation from a standard curveconstructed with sNRF plasmid DNA of known concentrations. Measurementswere performed in triplicate, with a deviation from the median value isless than 0.2%.

As shown in FIG. 5A, sNRF expression was significantly higher in theheart failure hearts compared to the control hearts in all cases. Also,NPRA gene expression had minimal variability (<2-fold change in allcases) and was not different when control and heart failure hearts werecompared (FIG. 5B) whereas significant differential expression wasobserved in sNRF expression in the heart failure hearts compared to thecontrol hearts (FIG. 5A).

Therefore, sNRF and NPRA are alternative products of the NPRA gene andsNRF (but not NPRA) is differentially regulated in human heart failure.

Similar methods can be used to measure sNRF in isolated humancardiomyocytes, and can be compared to CF sNRF levels. For example, RNAcan be extracted from the myocytes that remain in the supernatantfollowing the disassociation of the CF. Heart failure and CF and myocytesNRF gene expression can be compared to the levels in CF and myocytesobtained from recent post-mortem individuals with no cardiac history byNorthern blot.

Example 6 Determining Severity of Heart Failure with sNRF Expression

This example describes methods that can be used to correlate theseverity of clinical heart failure with sNRF mRNA expression. Asdescribed in Examples 4 and 5, quantitative RT-PCR can be used tomeasure sNRF expression in heart tissue. The information obtained fromheart failure patients (such as those described in Example 5), can beused to determine if levels of sNRF expression correlate with severityof heart failure.

Using SPSS software Version 14, analysis frequencies can be run oncategorical variables (NYHA class, etc.) and descriptive analysis oncontinuous variables in order to locate and isolate outliers. Based onthese results, transformations will be performed as appropriate. Next,sNRF expression can be compared to continuous variables (BNP, CRP, etc.)using independent t-tests and one-way ANOVAs. Chi-square will be used toassess independent categorical-type variables for co-linearity. Theprimary model will be a multivariate regression analysis using dummycoding for the independent categorical variables. Initial modeling willuse stepwise regression to define the best sNRF predictors. Variableswill be added one at a time using standard techniques (entry criterion:p value <0.10, exit criteria: p value >0.15). It is expected that NYHAclass, BNP level, and CRP will be among these.

If this analysis demonstrates that myocardial sNRF mRNA expressionlevels are predictive of heart failure risk or predictive of significantclinical end points such as heart transplantation or death, then alogistic regression model will be employed. Logistic regression willalso be used to explain confounding among the independent variables andto create associated odds ratios.

If there is a correlation between sNRF expression and severity of heartfailure, routine methods of measuring sNRF expression in clinicalsamples can be used to assess the severity of heart failure in apatient.

Example 7 PKG and NPRA Associate in Cardiac Fibroblasts

This example describes methods used to demonstrate that cGMP-dependentprotein kinase I (PKG) associates with natriuretic peptide receptor A(NPRA) in primary cardiac fibroblast (CF) cells.

A minimal PKG binding domain in NPRA was identified by expression ofsNRF (sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾(SEQ ID NO: 4), sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ (SEQ ID NO: 6) andsNRF⁽⁸⁰⁶⁻⁸⁶⁰⁾ (SEQ ID NO:8)) in transfected CFs and assayed theirability to block PKG-NPRA binding. None of the fragments containedeither the extracellular ligand-binding domain (amino acids 54-415) orthe kinase-like domain (amino acids 547-804) of NPRA. sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾includes both the an amphipathic helix (so-called hinge domain) and theC-terminal guanylyl cyclase domain. sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ excludes thecyclase-containing domain. sNRF⁽⁸⁰⁶⁻⁸⁶⁰⁾ contains only the hinge domain.

HEK-NPRA cells were transiently transfected with insertless pcDNA3plasmid vector or pcDNA3-sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾. All slide wells were treatedwith atrial natriuretic peptide (ANP) (100 nM; Sigma-Aldrich #A1663) for15 minutes. Cells were decorated with monoclonal flag antibody specificfor FLAG-NPRA (red) and polyclonal PKG antibody (green). Overlappingregions of PKG and NPRA appeared yellow-orange. PKG staining was absentwhen anti-PKG antibody was pre-adsorbed with recombinant PKG protein(Calbiochem). In control experiments, no immunofluorescence was seen inthe absence of primary antibody.

Expression of sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾ in HEK293 cells stably over-expressingFLAG-epitope tagged NPRA (HEK-NPRA cells) inhibited translocation ofcytosolic PKG to the plasma membrane, indicating that sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾interferes with PKG NPRA association. Although PKG-sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾co-localization could be readily seen in CFs, inhibition oftranslocation was more difficult to appreciate in these smaller cellsexpressing only endogenous NPRA. Therefore, expression of sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾blocks NP-induced PKG membrane translocation.

To demonstrate that PKG and NPRA interact in vivo, the ability of sNRFto immunoprecipitate PKG was determined in primary CF cells. Neonatalrat ventricular fibroblasts (CFs) were cultured from 1 to 2-day-oldHarlan Sprague-Dawley rats as described previously (Simpson, CirculationRes. 56:884-94, 1985). Briefly, ventricles were dissected free fromatria and quartered. Following dissociation in ADS buffer containing 1.5mg/ml trypsin (Gibco #27250-018) and 1% DNase solution (2 mg/ml DNasetype II, 150 mM NaCl) with serum neutralization the collected cells werepre-incubated in media at 37° C. for 90 minutes to allow CF attachmentto the bottom of culture dishes. CFs were incubated in plating media for48 hours, divided into 6-well culture plates or dual-well chamberslides,and grown to 50% confluency in DMEM with 10% FBS prior to use. Othershave shown that cells cultured in this fashion exhibit positive stainingfor vimentin, negative staining for von Willebrand factor, α-smoothmuscle actin, and sarcomeric actin, indicating that there is no relevantcontamination of the cardiac fibroblast culture with endothelial cells,smooth muscle cells, or cardiac myocytes (Tsuruda et al., Circ. Res.90:128-34, 2002).

CFs were transiently transfected with nucleic acid molecules encodingsNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾, sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾, or sNRF⁽⁸⁰⁶⁻⁸⁶⁰⁾ using routine methods.Briefly, PCR-generated, 5′-FLAG epitope-tagged sNRFs were subcloned intothe BamHI and EcoRI sites of pcDNA3 (Invitrogen) and sequenced to verifyconstruction accuracy. The following primer sequences were used togenerate 5′-FLAG epitope-tagged NPRA fragments: sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾):Forward: GGGTGGATCCACCATGGACTACAAAGACGATGACGACAAGAGGGAGAACAGCAGC AACAT(SEQ ID NO: 9), Reverse: CGGGAATTCTCAGCCTCGGGTGCTACTCC (SEQ ID NO: 10);sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾: Forward:GGGTGGATCCACCATGGACTACAAAGACGATGACGACAAGAGGGAGAACAGCAGC AACAT (SEQ IDNO: 9), Reverse: CGGGAATTCTGACAGGGTCACCACCTGCATGG (SEQ ID NO: 11);sNRF⁽⁸⁰⁶⁻⁸⁶⁰⁾: Forward:GGGTGGATCCACCATGGACTACAAAGACGATGACGACAAGAGGGAGAACAGCAGC AACAT (SEQ IDNO: 9), Reverse: CGGGAATTCTGACTCAGCCACTGAGTGAGGCA (SEQ ID NO: 12).

The ability of the NPRA fragments to immunoprecipitate PKG wasdetermined in the primary CF cells transfected with the 5′-FLAGepitope-tagged NPRA fragments as follows. CFs were collected andsolubilized in 1 ml ice-cold IP buffer containing 50 mM Hepes, pH 7.4, 5mM NaCl, 5 mM EDTA, 10% glycerol, 0.5% Triton X-100, and proteaseinhibitors (complete mini-tablet, Roche). Equal concentrations of lysatewere incubated with 20 μl anti-FLAG M2 affinity gel (Sigma # F-3165) for18 hours at 4° C. before 5 sequential 5-minute centrifugations with 1 mlfresh buffer before elution with hot SDS sample buffer (0.25 M Tris-HCL,pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.0025%bromophenol blue). Samples were boiled for 10 minutes and 30-μL aliquotsof eluate were loaded onto SDS-PAGE gels (5% acrylamide stacking phase,7.5% or 10% acrylamide resolving phase gels). Gels were transferred tonitrocellulose using a Bio-Rad Liquid Transfer Unit in transfer bufferwith 10% methanol. Before blocking (5% nonfat dry milk, TBS, 0.03%Tween) and incubation with primary antibody (Santa Cruz Biotechnology)overnight at 4° C. Blots were then washed 3 times for 5 minutes withTBST before incubation with a 1:5000 dilution of horseradishperoxidase-conjugated donkey anti-IgG antibody (Jackson Laboratories) inTBST. Bound antibody was detected with chemiluminescent substrate(Western Lightning ECL, PerkinElmer Life Sciences).

sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾ and sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾, but not sNRF⁽⁸⁰⁶⁻⁸⁶⁰⁾,co-immunoprecipitated PKG, indicating that both sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾ andsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ can bind PKG and potentially compete with endogenous NPRAfor PKG binding. sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ failed to associate with full-lengthNPRA, either in the presence or absence of activated PKG, indicatingthat the fragment's effects are not based on its ability to dimerizewith the full-length receptor.

Therefore, sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾, the shortest fragment observed to associatewith PKG, was used to demonstrate the effect of PKG-NPRA association ondownstream actions, receptor cyclase activity, and receptorphosphorylation.

Example 8 PKG Phosphorylation of NPRA in Inhibited by sNRF

This example describes methods used to demonstrate that PKG canphosphorylate NPRA, and that this phosphorylation is inhibited by sNRF.

Phosphorylation of specific serine and threonine residues within theNPRA kinase homology domain is required for ANP-induced action. Forexample, it has previously been shown that deletion of the kinasehomology domain itself results in ligand-independent, constitutivereceptor guanylyl cyclase activity (Wedel et al., Proc. Nat. Acad. Sci.USA 94:459-62, 1997; Chinkers et al., Science. 245:1392-4, 1989).

To confirm that PKG, a serine-threonine kinase, is capable ofphosphorylating NPRA, the following methods were used. Cos 7 cells weretransfected with a cDNA vector encoding full-length NPRA (SEQ ID NO: 2)or with an empty vector, and treated with PKG (20 U). Lysates preparedfrom the cell membranes of Cos 7 cells were treated with recombinant PKGand then divided into two aliquots. One aliquot was incubated (30minutes) with 8-bromo-cGMP and [³⁵S] ATPγS and then separated bySDS-PAGE, blotted to Immobilon membranes, and visualized byautoradiography. The other aliquot was separated similarly and thenprobed with anti-NPRA antisera (ABCAM ab14356).

As shown in FIG. 6A, SDS page gels demonstrated a phosphorylated 137 kDprotein that corresponded to full-length NPRA.

To demonstrate that PKG phosphorylates NPRA in an ANP-dependent manner,CF cells were transfected with either insertless vector or sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾(see Example 7 and FIGS. 6C and 6D) and treated with or without ANP for5 minutes in the presence or absence of 1 μM of the PKG inhibitor KT5823(Alexis Biochemicals, Cat #270-087-C100). To identify receptorphosphorylation by PKG, [³⁵S]ATPγS was employed as a substrate. [³⁵S]incorporation into NPRA was analyzed according to the method previouslydescribed (Joubert et al., Biochem. 40:11096-105, 2001). Briefly, thetreated CFs were scraped into 100 μl ice-cold incubation buffer (25 mMHEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl₂, 0.2 μM okadaic acid, 1 mM Na₃VO₄,10 mM NaH₂PO₄, 10 mM NaF, and protease inhibitors (complete mini-tablet,Roche) and lysed by repeated passage through a 22-gauge needle. Lysateswere added to incubation buffer containing 1 μCi [³⁵S]ATPγS (specificactivity >1000 Ci/mmol), final volume of 200 μl, incubated at 37° C. for30 minutes. Proteins were analyzed by SDS-PAGE (7.5%) and subsequentautoradiography.

Since the lysates used in the kinase reaction also contained an array ofprotein phosphatase inhibitors, the possibility of reduced phosphataseactivity as a mechanism for receptor phosphorylation or increasedphosphatase activity as a major mechanism sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾-inducedinhibition phosphorylation was excluded. In other control experiments,PKG treatment resulted in phosphorylation of whole cell lysatepreparations from HEK-NPRA cells, but not from cells expressing littleor no NPRA. As shown in FIGS. 6B-5D, both KT5823 or expressedsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ protein reduced ANP-induced NPRA phosphorylation tocontrol levels.

To ensure that the 137-kD phosphorylation band observed in the CFs afterANP treatment was, in fact, NPRA, HEK293 or HEK-NPRA were treated withANP with or without KT5823 as described above. The lysates were dividedinto two aliquots. One aliquot was incubated with [³⁵S] ATPγS and thenseparated by SDS-PAGE, blotted to Immobilon membranes, and visualized byautoradiography. The other aliquot was separated similarly and thenprobed with anti-NPRA antisera, stripped and re-probed withanti-α-tubulin to assure equal protein loading. Robust phosphorylationof a 137-kD protein occurred in the NPRA-expressing cells, but not incontrol cells (FIG. 6B). Since both KT5823 and sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾ inhibitcytosolic-to-plasma membrane translocation of PKG, PKG-NPRA associationappears to be required for PKG-induced NPRA phosphorylation.

Therefore, expression of sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ results in ligand-independent,constitutive cGMP generation, and PKG phosphorylation of NPRA isinhibited by sNRF. The earlier observation, that the elimination of PKGcatalytic activity by KT5823 inhibits PKG translocation (Airhart et al.,J. Biol. Chem. 278:38693-8, 2003), and the current observation that bothKT5823 and sNRF protein expression blocks both basal and NP-inducedreceptor phosphorylation, indicate that PKG's kinase activity isinvolved in regulating receptor responsiveness. Based on these data, amodel is proposed in which basal PKG-NPRA association and NPRAphosphorylation allows ligand-induced PKG translocation and receptorphosphorylation. Subsequently, NP-induced further accumulation of PKGand increased NPRA phosphorylation, in turn, amplifies cyclase activity.

In summary, these results demonstrate that the association of PKG withNPRA modulates NPRA responsiveness to NPs through PKG's NPRA kinaseactivity in native cardiac cells expressing only endogenous PKG andNPRA. A sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ fragment (SEQ ID NO: 6) that does not containNPRA's extracellular ligand-binding or intracellular kinase homologydomains, but does contain the hinge and a small 5′ portion of theC-terminal guanylyl cyclase domain, associates either directly orindirectly with PKG. By sequestering PKG from its NPRA binding site,sNRF attenuates PKG's function as an adaptor or bridging protein thathas a permissive effect on ANP-induced conformational changes in NPRAand dimerization of its C-terminal cyclase domains.

These results demonstrate that PKG potently phosphorylates NPRA in anNP-dependent manner, and that a blocker of PKG activation, KT-5823,inhibited this effect. Furthermore, sNRF expression inhibits NP-inducedNPRA phosphorylation. Since sNRF binds PKG and also blocks NP-dependentPKG translocation, sNRF inhibits NPRA phosphorylation and function byaltering PKG's interaction with NPRA. These findings indicate that PKGplays a role in the regulation of receptor responsiveness

Based on these observations, methods of (1) decreasing biologicalactivity of sNRF to treat heart failure and (2) screening compounds fortheir ability to enhance the sensitivity of an NPR for NP ligands, aredisclosed.

Example 9 Effect of PKG-NPRA Association on NPRA Catalytic Activity

This example describes methods used to demonstrate that expression ofsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ significantly reduces CFs response to ANP.

The ability of sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ to reduce or inhibit downstream effects bymodulating the intrinsic guanylyl cyclase activity of NPRA, wasdetermined by measuring total cGMP levels in NP-treated CFs. CFs weretransfected with insertless vector or sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ as described inExample 7 and treated with ANP (100 nM) for 15 minutes. cGMPconcentration was determined by enzyme immunoassay using a commercialkit (Enzyme immunoassay Biotrak EIA System, Amersham Biosciences). Inall experiments, cells were treated with 100 μM IBMX with or withoutANP.

As shown in FIG. 7, ANP treatment significantly increased intracellularcGMP concentration in CF cells. However, sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ alone alsosignificantly increased cGMP levels. This effect was not due to aneffect on phosphosdiesterase activity, because all experiments wereconducted in the presence of the non-selective phosphodiesteraseinhibitor, IBMX. Furthermore, since sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ does not appear todirectly associate with the full-length receptor, and since thisfragment lacks the guanylyl cyclase domain, it is unlikely that thiseffect is due to sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ functioning as a dominant negative. ANPtreatment of sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾-expressing cells did not lead to anadditional increase in cGMP concentration, compared to the effect ofsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ alone (FIG. 7).

Therefore, sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ expression in CFs leads to constitutivereceptor activation and resistance to further stimulation by ANP, byisolating PKG from its NPRA binding sites. This indicates thatexpression of sNRF mimics desensitization of NPRA for ANP ligand thatoccurs during heart failure.

Example 10 sNRf Blocks NP Inhibition Of bFGF-Induced Cardiac Fibrosis

This example describes methods used to demonstrate that the associationof PKG with sNRF influences NPRA-mediated signaling, and that sNRFreverses ANP inhibition of bFGF-induced α-SMA expression.

The degree of CF-induced myocardial fibrosis caused by their elaborationof collagen and extracellular matrix after myocardial infarction andduring cardiac remodeling largely determines the outcome of clinicalheart failure. When animals are stressed by experimental myocardialinfarction, or when CFs are grown in tissue culture and treated withgrowth factors such as TGF-β₁ or bFGF, they differentiate intomyofibroblasts, begin to express NPs, and produce significant amounts ofα-SMA. NP treatment of growth factor-stimulated CFs inhibits theirproliferation and expression of collagen, extracellular matrix, andα-SMA. Thus, CFs are a model in which to determine the downstreameffects of NPRA activation because they express endogenous NPRA and PKG,their NP system is closely regulated as cardiac disease develops, and,in general, CFs play a role in the development of cardiac disease.

NP's ability to inhibit growth factor-induced CF differentiation intomyofibroblasts was determined as follows. CFs were transientlytransfected with insertless pcDNA3 vector or pcDNA3 encodingsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ (see Example 7). The transfected CFs were then treated for48 hours with 10 ng/ml basic fibroblast growth factor (FGF;Sigma-Aldrich # F0291) in the presence or absence of 100 nM ANP.Alpha-smooth muscle actin (α-SMA) protein expression, the principalmarker of CF differentiation, was assessed by Western blotting andimmunofluorescence microscopy. Vimentin staining was used as a loadingcontrol because it is expressed equally in fibroblasts andtransdifferentiated myofibroblasts.

For the immunofluorescence microscopy, slide wells were treated asdescribed in Example 7 and incubated with anti-α-SMA and anti-vimentinantibody as well as phalloidin to stain for actin. Briefly, cells wererinsed twice with phosphate-buffered saline (PBS) and fixed with 3.7%formaldehyde in PBS for 10 minutes. After fixation, cells were rinsedwith PBS and permeabilized with 0.3% Triton X-100 in PBS for 10 minutesand blocked for 1 hour in 1% horse serum, 0.2% bovine serum albumin inPBS. Cells were then incubated with primary α-SMA antibody (ABCAMab7817) and primary vimentin antibody (ABCAM ab7783) diluted in blockingsolution for 1 hour and washed three times with blocking solution for 5minutes. The cells were incubated with the appropriate fluorescentsecondary antibodies for 1 hour (FITC and Cy5 conjugated-secondarydonkey antibodies; Jackson Laboratories) and a 1:1000 dilution ofrhodamine-phalloidin (Molecular Probes), which was used to visualizeactin fibers. After three PBS washes, the cells were mounted on glasscoverslips using Slowfade Antifade mounting medium (Molecular Probes).Images were acquired using a Leica DMRA Fluorescent Microscope equippedwith a 40×/0.7 HCX PL Fluotar objective, Leica N3, Y5, and L5 filtercubes, a Hamamatsu Ocra-ER digital camera, and Openlab 3 software.

As shown in FIGS. 8A and 8B, treatment of CFs with growth factorsignificantly increased α-SMA protein expression in CFs. ANP treatmentinhibited bFGF-induced α-SMA protein expression in cells transfectedwith insertless vector (FIGS. 8A, 8B). However, expression ofsNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾ reversed ANP's inhibitory effect on growth factor-inducedα-SMA protein expression. While the Western immunoblots demonstratedequal amounts of vimentin in all treatment groups, immunofluorescencemicroscopy indicated that control and FGF-treated cells had diffusenuclear and cytosolic staining, whereas nuclear to cytosolictranslocation of vimentin was observed in the presence of ANP, an effectthat was inhibited by expression of sNRF⁽⁸⁰⁶⁻⁹⁰⁰⁾.

Therefore, association between PKG and an intracellular domain of theNPRA C-terminus is needed for NP's inhibitory action on myofibroblastdifferentiation. The region on PKG that binds to NPRA is referred to theNPRA association domain on PKG (NAD), and the region on NPRA binds toPKG is referred to the PKG association domain on NPRA (PAD).

Example 11 sNRF Blocks NP Inhibition of TGF-β₁-Induced Cardiac Fibrosis

This example describes methods used to demonstrate that sNRF reversesANP inhibition of TGF-β₁-induced α-SMA expression.

TGF-β₁ signaling is required for angiotensin effects in the heart, andNPs oppose TGF-β₁ effects in CFs. To demonstrate the effect of sNRFexpression on TGF-β₁-induced α-SMA luciferase reporter expression, andto determine if common heart failure therapies (such as (3-adrenergicblockade (propranolol), angiotensin-converting enzyme inhibition(captopril), or angiotensin receptor 1 blockade (losartan)), reversedsNRF effects, the following methods were used. CFs were transientlytransfected with the α-SMA-luciferase vector and pcDNA3 control vectoror pcDNA3-sNRF, then treated for 24 hours with TGF-β₁, ANP, and theangiotensin-converting enzyme inhibitor captopril (CAP) or theangiotensin receptor-1 blocker losartan (LOS).

As shown in FIG. 9, sNRF expression in untreated CFs significantlyincreased α-SMA-luciferase expression compared to non-sNRF expressinguntreated cells. Furthermore, sNRF expression in TGF-β₁-treated cellsincreases α-SMA luciferase expression more than four-fold compared toTGF-β₁-treated control cells. sNRF expression significantly amplifiedreporter induction in both control and TGF-β₁-treated cells. NPtreatment inhibits TGF-β₁-induced α-SMA-luciferase expression in cellsthat do not over-express sNRF (FIGS. 1 and 7). However, sNRFover-expression abolishes NPs' inhibitory effect. Angiotensin inhibitionwith either captopril or losartan had no significant inhibitory effecton this sNRF action (FIG. 7). In fact, in non-sNRF transfected cells,losartan appears to have a sNRF-like effect (losartan blocksNP-inhibition of TGF-β₁ induction of α-SMA-luciferase). β-adrenergicblockade also had no effect on sNRF action.

In summary, standard heart failure therapies appear to have little or noinhibitory effect on sNRF action. These data indicate that β-blockade orangiotensin inhibition will not be effective therapies against sNRF'sdeleterious action.

Example 12 Reducing the Biological Activity of sNRF

This example describes methods that can be used to reduce the biologicalactivity of sNRF as a method of treating heart disease. Based on theobservations described in Examples 4 and 5 (FIGS. A-D and 5A-B) showingthat sNRF mRNA expression is elevated in heart disease, sNRF is a targetfor heart failure therapies designed to reduce or block sNRF action.Although particular methods of reducing sNRF activity by blockingexpression with siRNA are described, one skilled in the art willappreciate that similar methods can be used with other agents thatsignificantly decrease sNRF expression or activity, such as otherinhibitory nucleic acid molecules.

As shown in the Examples above, sNRF expression inhibits NPs' beneficialactions, such as its ability to block the growth factor-induceddifferentiation of cardiac fibroblasts to myofibroblasts. The methods inthis example provide methods of reducing sNRF expression using siRNA,thereby restoring NPs' beneficial effects in NP-resistant cells.

Primary fibroblast cultures can be obtained from human tissue derivedfrom explanted heart from patients prior to orthotopic hearttransplantation. As an alternative, human CFs can be purchased fromScienCell Research Laboratories (San Diego, Calif.). Non-cardiac failurehuman fibroblasts can be derived from discarded material from childrenand adults undergoing cardiac surgery. Post-mortem tissue fromindividuals without a cardiac history can also be obtained as a sourceof CFs. An additional source of heart tissue is myocardial plugsdiscarded after patients are placed on mechanical ventricular assist.

The neonatal rat CF culture protocol (Simpson, Circ. Res. 56(6):884-894,1985), modified for human cell culture according to the method of Agochaet al. (Cell Tissue Res. 288(1):87-93, 1997), can be used to cultureprimary CF cells. The heart muscle is immediately minced and placed inADS buffer containing 1.5 mg/ml trypsin (Gibco cat. #27250-018),collagenase (type IV, Sigma) and 1% DNase solution (2 mg/ml DNase typeII in 150 mM NaCl) with serum neutralization. The collected cells arepre-incubated in media at 37° C. for 90 minutes to allow CF attachmentto the bottom of culture dishes. Myocytes in the supernatant and aportion of the CFs are placed in RNA stat-60 (Iso-Tex Diagnostics, Inc.)for subsequent sNRF mRNA quantitation. The remaining CFs are eitherflash-frozen in liquid nitrogen and placed in −80° C. for future use, orincubated in plating media for 48 hours, divided into multi-well cultureplates and grown to 50% confluency in DMEM with 10% FBS prior totransfection and/or subsequent experimental protocols. These cells canbe used effectively for up to 9 passages. Experiments will be performedon passage 2 or 3 cells. To confirm that there is no relevantcontamination of fibroblasts with endothelial cells, smooth musclecells, or cardiac myocytes, cells will be stained for vimentin, for theabsence of von Willebrand factor, α-SMA, and sarcomeric actin.

Small interfering (si)RNA molecules specific for sNRF can be generatedas follows. The sNRF cDNA clone (GenBank Accession No. BX329044)contains a 215-nt sequence corresponding to intron 15 of the genomicNPRA sequence (nucleotides 1-215 of SEQ ID NO: 37) that is not presentin the native NPRA cDNA. This sequence, therefore, presents a uniquetarget for siRNA-directed sNRF knockdown that should have no substantialeffect on full-length NPRA expression. Since single siRNA sequences mayeach have off-target effects or varying degrees of silencing, least twoeffective sNRF siRNAs, including “ON-TARGET plus” modified siRNAsthought to decrease off-target effects up to 90% (Dharmacon) are used.Sequences at the 5′ end of the presumed sNRF 5′-UTR are targeted so asto avoid the intron 15-exon 16 splice acceptor site and the adjacent 5′branch point (usually ˜35 nt upstream of the 3′ end of the relevantintron), whose interaction with a sNRF-directed siRNA could modulateexpression of the full-length NPRA mRNA. As a functional control forthis possibility, quantitative RT-PCR can be used to measure native NPRAmRNA using a probe that is complementary to mRNA 5′ of the sNRFsequence. In addition to specific control siRNAs, scrambled sNRF oligosor GFP siRNA can be used.

Particular exemplary siRNA molecules that can be used to silence sNRFexpression include those shown in SEQ ID NO: 14, 16, 18, 20, 22, 24, 26,28, 30 or 32 (as well as duplexes of these hybridized to theircomplementary sequences, such as those sequences shown in SEQ ID NO: 13,15, 17, 19, 21, 23, 25, 27, 29, and 31, respectively).

To achieve high transfection efficiency, the transfection efficiency ofseveral transient transfection reagents (HiPerFect, Quiagen; GeneEraser,Stratagene; FuGene 6, Roche; Lipofectin, Invitrogen) can be determined.Transfection efficiency can be determined using GAPDH siRNA (Dharmacon)and subsequent quantification of GAPDH mRNA.

Titration curves using each of the sNRF siRNA molecules can beconstructed, since off-target effects are minimized at low siRNAconcentrations. The titration curve can be constructed based onquantitative RT-PCR determinations of sNRF mRNA expression. ThesNRF-specific primers shown in FIG. 2 (“a” and “b”) will be use. Asdescribed above, two different siRNA sequences that produce at least a50% reduction of sNRF mRNA expression when using at a maximum of 40-50nM will be employed for subsequent methods.

Both commercial and patient-derived cells will be analyzed for α-SMAprotein using Western blots probed with specific polyclonal antibody(FIG. 9) and sNRF mRNA expression. Untreated cells will likely have lowlevels of α-SMA. If fibroblasts cultured from failing myocardiumcontained increased α-SMA compared to cells derived from non-failinghearts, this would indicate their transdifferentiation intomyofibroblasts. This is not impossible, since injured myocardium hasincreased α-SMA expression. It is more likely, however, that culturedfibroblasts will de-differentiate and express low amounts of α-SMA thatare typical of unstimulated cells.

As shown in the Examples above, failing myocardium has elevated sNRFmRNA levels. This will be confirmed in cultured fibroblasts derived fromfailing heart compared to non-failing fibroblasts, as well as forcardiomyocytes in the supernatants obtained during CF tissue culture.

Using the methods described in Example 1, human CFs will be transfectedwith the α-SMA-luciferase vector with or without sNRF siRNA and treatedwith TGF-β₁ with or without NP, for up to 4 days. It is expected that atime-dependent increase in α-SMA-luciferase expression and sNRF mRNAconcentration in the TGF-β₁+NP-treated cells, but not in thesiRNA-transfected TGF-β₁+NP-treated cells, will be observed. This resultwould demonstrate that NP resistance is caused by sNRF.

To confirm this observation, and as an independent verification of thespecificity of the siRNAs used, one of two pcDNA3 plasmid vectors thatcontain either 1) the sNRF open reading frame-alone or 2) a mutated sNRF5′-UTR/intron-15 sequence that is not complementary to the effectivesNRF siRNA(s) will be co-transfected. Since the sNRF siRNA targets thesNRF 5′-UTR (encoded by intron 15 of the NPRA gene), co-transfections ofthe sNRF plasmid (containing only the open reading frame) or the mutatedsequence should both rescue the ability of prolonged NP exposure todesensitize NPRA. In the first rescue, prolonged NP exposure will resultin progressively increased α-SMA-luciferase expression. In the secondrescue, it is expected that over the 4-day period increasingα-SMA-luciferase expression and increasing sNRF mRNA levels inquantitative RT-PCR experiments will be observed.

If efficient sNRF siRNA oligomers cannot be designed from the 215-ntNPRA intron-15 sequence (nucleotides 1-215 of SEQ ID NO: 37), rapidamplification of 5′ complementary DNA ends (5′ RACE) can be used todelineate the 5′ end of the sNRF mRNA. This will provide additional 5′sequence information, to which additional sNRF-specific siRNAs can betargeted using routine methods known in the art.

siRNA or other inhibitory molecules shown to be effective in vitro canbe further tested in vivo, for example in a laboratory animal model forheart failure (see Example 16).

Example 13 Effect of sNRF on the TGF-β₁ Signaling Pathway

TGF-β₁ signaling plays a role in the development of cardiac hypertrophyand heart failure, and sNRF potentiates TGF-β₁'s ability to inducecardiac fibroblast differentiation. This example describes methods thancan be used to determine whether this effect is due to inhibition ofbasal NPRA activity or through independent regulation of TGF-β₁signaling itself, using NPRA-expressing and NPRA-deficient cells.Similar methods can be used to screen for compounds that decrease thesNRF-induced deleterious growth factor effects (such as TGF-β₁'s abilityto induce cardiac fibroblast differentiation).

One explanation for the observation that sNRF potentiates TGF-β₁activity is that sNRF-PKG association inhibits PKG action (perhaps byinhibiting nuclear translocation of PKG), thereby preventing PKGinhibition of thrombospondin transcription. As shown in FIG. 9, sNRFexpression increases basal α-SMA expression and also significantlyincreases TGF-β₁-induced α-SMA expression compared tonon-sNRF-expressing, untreated CFs. This example provides methods thatcan be used to determine if sNRF's TGF-β₁-potentiating effect is due toinhibition of basal NPRA activity or through a direct effect on theTGF-β₁ signaling system.

Commercial or patient-derived human CFs will be used as described in theExamples above. To determine if sNRF's potentiation of TGF-β₁ signalingis a direct effect that is independent of NPRA, NPRA gene expressionwill be decreased using NPRA siRNA. Multiple siRNA sequences can beevaluated (such as the exemplary siRNA duplex molecules shown in SEQ IDNOS: 13 and 14, SEQ ID NOS: 15 and 16, SEQ ID NOS: 17 and 18, SEQ IDNOS: 19 and 20, and SEQ ID NOS: 21 and 22). Transfection efficiencyusing GAPDH siRNA and optimal concentration will be determined asdescribed in Example 12. NPRA protein expression will be assessed byWestern immunoblotting using NPRA-specific antisera (ABCAM ab14356), andNPRA mRNA expression will be measured by quantitative RT-PCR.

NPRA-deficient cells are transfected with pcDNA3-sNRF or control vectorbefore measurement of receptor activation. TGF-β₁ receptors initiateintracellular signal transduction by phosphorylation of members of theSmad protein family. Phospho-Smads 2 and 3 initiate transcriptionalevents after nuclear translocation. Therefore, following NPRA knockdownin pcDNA-sNRF-transfected cells, TGF-β₁-induced α-SMA luciferase andprotein expression will be compared to cells expressing normal NPRAlevels in addition to Smad phosphorylation and nuclear translocation.

In cells over-expressing sNRF, increased TGF-β₁-induced α-SMA expressionin the presence of NPRA knockdown would support the conclusion that sNRFaction is NPRA-independent. If this effect were due specifically toincreased TGF-β₁ signaling, then Western immunoblots probed withanti-phospho-Smad (anti-Smad2—Signal Transduction Labs andanti-phospho-Smad2-Upstate Biotech.) would show increased Smadactivation compared to cells not over-expressing sNRF.

If sNRF over-expression increases TGF-β₁ signaling through binding PKGand blocking PKG's inhibition of thrombospondin transcription, then sNRFover-expression in NPRA-silenced cells treated with TGF-β₁ may decreaseintranuclear PKG observed using immunofluorescence microscopy (methoddescribed in Example 7) or decreased phosphorylation activity asdemonstrated by in vitro PKG kinase activity assays. It is expected toobserve increased thrombospondin mRNA and protein expression(anti-thrombospondin antibody, Abcam #ab2962) in sNRF over-expressingcells. The opposite would occur in sNRF-silenced and NPRA-silenced cellstreated with TGF-β₁ (that is, increased nuclear PKG, increased in vitroPKG kinase activity, and decreased thrombospondin).

Example 14 Identification of Therapeutic Agents

This example provides a particular example of an in vitro assay that canbe used to screen agents for their potential to treat a cardiovasculardisorder, such as heart failure. For example, agents can be screened fortheir ability to increase the biological activity of NPRA/B, inhibit thebiological activity of sNRF, decrease sNRF-induced deleterious growthfactor effects (such as TGF-β₁'s ability to induce cardiac fibroblastdifferentiation) or combinations thereof, for example by increasing thesensitivity of NPRA/B to NPs. Agents identified via the disclosed assayscan be useful, for example, in restoring or enhancing the beneficialeffects of NPs, for example in treating a subject having a disease thatresults from decreased biological activity of NPR that results when thereceptor becomes desensitized to the presence of NPs, such ascardiovascular disease. In addition, agents identified via the disclosedassays can be useful, for example, in decreasing deleterious growthfactor effects, for example in treating a subject having a disease thatresults from sNRF-induced deleterious growth factor effects, such ascardiovascular disease.

Although particular examples are provided for screening, one skilled inthe art will appreciate that variations to the disclosed method can bemade, without affecting the assay. For example, other growth factorsbesides FGF or TGF-β can be used (such as epidermal growth factor (EGF),fibroblast growth factor (FGF), erythropoietin (EPO), growth hormone(GH), insulin-like growth factor, insulin, hematopoietic cell growthfactor (HCGF), platelet-derived growth factor (PDGF), and vascularendothelial growth factor (VEGF)). In addition, other intracellular NPRAfragments can be used, such as sNRF⁽⁸⁰⁶⁻¹⁰⁶¹⁾ (SEQ ID NO: 4),sNRF⁽⁸²⁰⁻¹⁰⁶¹⁾ (SEQ ID NO: 40), or sNRF⁽⁸²⁰⁻⁹⁰⁰⁾ (SEQ ID NO: 42).

As disclosed in the examples above, intracellular NPRA fragments (sNRF)that interfere with the association or binding between PKG and NPRAmimic the physiology of heart failure, for example by increasing cGMPlevels in CFs. Therefore, screening assays that utilize sNRF can be usedto identify and analyze agents for their ability to increase thebiological activity of NPR or to decrease the biological activity ofsNRF. In particular examples, agents identified via the disclosed assayscan be useful in increasing the sensitivity of a desensitized NPR toNPs, for example in treating a subject having (or at risk fordeveloping) cardiovascular disease. Assays for testing the effectivenessof the identified agents, are discussed below.

It is also disclosed in the examples above, that sNRF potentiatesdeleterious growth factor effects, such as cardiac fibroblastdifferentiation and expression of collagen, extracellular matrix, andα-SMA. Therefore, screening assays that utilize sNRF can be used toidentify and analyze agents for their ability to decrease the biologicalactivity of sNRF. In particular examples, agents identified via thedisclosed assays can be useful in decreasing sNRF-induced potentiationof growth factor effects on cardiovascular disease, for example intreating a subject having (or at risk for developing) cardiovasculardisease. Assays for testing the effectiveness of the identified agents,are discussed below.

Exemplary agents that can be screened include, but are not limited to,any peptide or non-peptide composition in a purified or non-purifiedform, such as peptides made of D- and/or L-configuration amino acids (m,for example, the form of random peptide libraries; see Lam et al.,Nature 354:82-4, 1991), phosphopeptides (such as in the form of randomor partially degenerate, directed phosphopeptide libraries; see, forexample, Songyang et al., Cell 72:767-78, 1993), antibodies, nucleicacid molecules (such as RNAi molecules, for example siRNAs) and small orlarge organic or inorganic molecules. A test agent can also include acomplex mixture or “cocktail” of molecules.

Briefly, in one example the method includes cellular expression ofα-SMA-luciferase and sNRF in neonatal rat cardiac fibroblasts(sNRF-o-blasts). A TGF-β₁-induced signal is quenched after NP treatmentof primary cultures of control cells (FIG. 9). In this sNRF-o-blastsystem, sNRF expression reverses NP-induced inhibition of the reportersignal, a model that mimics NP resistance in human heart failure. Usingthis assay in transiently transfected cells, a library of smallmolecules can be screened for lead drugs that restore NP-inducedinhibition of TGF-β₁-stimulated luciferase expression.

Test agents can be screened for their ability to reverse the effects ofrecombinant sNRF molecules that interfere with the PKG-NPRA association.Primary CF cells are prepared using routine methods, for example asdescribed in Example 7. However, one skilled in the art will recognizethat other CF cells can be used, and other methods of preparing CF cellscan be used. The CF cells are transfected with a vector that includes anα-SMA promoter operably linked to a reporter sequence. α-SMA promotersequences are known. The α-SMA promoter sequence can be introducedupstream of luciferase, using the p-luc vector that includes thatluciferase reporter sequence (for example see Liu et al., J. Biol. Chem.278:48004-11, 2003). The resulting vector is transfected into CF cellsusing routine methods.

In addition, the CF cells include a recombinant sNRF protein that canbind PKG, such as those shown in SEQ ID NOS: 4, 6, 38, 40 or 42. sNRFcan include other elements, such as a FLAG tag. In one example, sNRF(such as SEQ ID NO: 4, 6, 38, 40 or 42) is operably linked to apromoter, and can be part of a vector. In a particular example, onevector is used that includes both the promoter-sNRF and α-SMApromoter-luciferase sequences.

CF cells that include a recombinant sNRF that can bind PKG and arecombinant α-SMA promoter sequence operably linked to luciferase (orother reporter sequence), are incubated with 10 ng/ml FGF or 5 ng/mlTGF-β, and 100 nM ANP for 15 minutes at 37° C. Simultaneously, before,or after incubation with ANP, cells are contacted with one or more testagents. In particular examples, the CF cells are present in a multi-wellplate, such as a 12-, 24-, 96-, 384-, or 1536-well plate. Such platingpermits high-throughput screening.

Particular examples of controls that can be included are:

-   -   (1) CF cells that include a recombinant α-SMA promoter sequence        operably linked to luciferase (or other reporter sequence)        incubated with growth factor, but not ANP or the test agent.        Such conditions should result in a positive signal from the        reporter.    -   (2) CF cells that include a recombinant α-SMA promoter sequence        operably linked to luciferase (or other reporter sequence)        incubated with growth factor and ANP, but not the test agent.        Such conditions should result in a negative signal from the        reporter.    -   (3) CF cells that include a recombinant sNRF that can bind PKG        (such as SEQ ID NO: 4, 6, 38, 40 or 42) and a recombinant α-SMA        promoter sequence operably linked to luciferase (or other        reporter sequence) incubated with growth factor and ANP, but not        the test agent. Such conditions should result in a positive        signal from the reporter.

Following incubation with the desired agents, the signal generated fromthe reporter is measured. For example, the luciferace signal generatedfrom the cells can be detected using a spectrophotometer. Detectableluciferase (or other reporter) signal should be detected in controls (1)and (3). However, decreased or even undetectable luciferase (or otherreporter) signal should be observed in control (2).

Test agents having similar luciferase (or other reporter) signal to thatin controls (1) and (3) are not expected to enhance the sensitivity ofNPR for NP ligands. In contrast, test agents having similar luciferase(or other reporter) signal to that in control (2), or that havesignificantly less than controls (1) and (3), may have the ability toenhance the sensitivity of NPR for NP ligands, and thus may be able totreat cardiovascular disease. Such agents can be selected for furtherexamination. For example, test agents identified using this assay can bescreened for their ability to bind PKG (see Example 7) or decreasesNRF-induced deleterious growth factor effects (for example see Example10 and 13), and for their ability to treat cardiovascular disease in ananimal model (see Example 16). Furthermore, such agents can be used totreat a subject having cardiovascular disease (see Example 18).

Example 15 Molecules for Disruption of sNRF Expression

This example describes siRNA, antisense, ribozyme, microRNA, and triplehelix molecules that can be used to reduce or disrupt expression ofsNRF, thereby decreasing the biological activity of sNRF. Such agentsare useful for treating heart failure, for example preventing heartfailure in a subject having increased risk for developing heart failure.Techniques for the production and use of such molecules are well knownto those of skill in the art. For example, nucleic acid sequences cansynthesized by use of an automated DNA synthesizer. Methods for usingthese molecules are described in Example 18.

The amount of siRNA, antisense, ribozyme, microRNA, or triple helixmolecule that is effective in the treatment of a particular heartdisease or condition (the therapeutically effective amount) depends onthe nature of the disease or condition, and can be determined bystandard clinical techniques. For example, it can be useful to usecompositions to achieve sustained release of such nucleic acidmolecules. In another example, liposomes containing the desiredtherapeutic molecule are targeted via antibodies to specific cells.

In particular examples, at least one of the particularly disclosed siRNAmolecules (SEQ ID NOS: 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32), aloneor hybridized to its respective sense sequence (SEQ ID NOS: 15, 17, 19,21, 23, 25, 27, 29, and 31, respectively, wherein the hybridized duplexis administered), or combinations thereof, is administered to a subjectto treat heart failure. In one example, the amount of disclosed siRNA,antisense, or ribozyme RNA administered (for example in a single dose)is 1-10 mg nucleic acid molecule/kg of subject, such as 1-5 mg/kg, or3-7 mg/kg. In particular examples, the inhibitory RNA molecule isadministered intravenously. In some example, the inhibitory RNA moleculeis delivered to a cell, such as a cell in a subject, using a viralvector. If desired, cells can be transfected with the desired inhibitoryRNA molecule, and the cells subsequently delivered to the subject usingmethods known in the art.

siRNA Molecules

siRNA molecules can be used to decrease or inhibit expression of sNRF,for example to decrease the ability of a pathogen to infect a cell, suchas infection by an enveloped RNA virus. Exemplary siRNA compounds areprovided herein, such as the exemplary siRNA antisense molecules shownin SEQ ID NOS: 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32. One skilled inthe art will appreciate that these antisense molecules can by hybridizedto their sense strand (SEQ ID NOS: 15, 17, 19, 21, 23, 25, 27, 29, and31, respectively), and the resulting duplex used as the therapeuticmolecule. However, the disclosure is not limited to these particularsiRNA molecules. Based on the disclosed sNRF sequences (such as intron15 of NPRA, nucleotides 1-215 of SEQ ID NO: 37), one skilled in the artcan generate other siRNA molecules using known methods. For example,siRNA sequences that recognize sNRF sequences can be designed andprepared by commercial entities, such as Sequitur, Inc. (Natick, Mass.)and Dharmacon (Lafayette, Colo.).

This disclosure is not limited to siRNA compounds of a particularlength. A siRNA molecule specific for sNRF can be any length, such as atleast 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides,at least 22 nucleotides, at least 23 nucleotides, at least 24nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least27 nucleotides, or at least 30 nucleotides.

Using the methods described herein (for example, see Example 18), siRNAcompounds can be used to treat heart failure, prevent future heartfailure, or combinations thereof. For example, an siRNA compound shownin any of SEQ ID NOS: 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32 isincubated with its reverse complement (SEQ ID NOS: 15, 17, 19, 21, 23,25, 27, 29, and 31, respectively), allowing hybridization of the twomolecules. In particular examples, two or more, such as three or more,or four or more, siRNA compounds are introduced into a cell. Forexample, the duplex molecule is contacted with a cell, such as a cell ofa subject in whom relief of symptoms associated with heart failure isdesired, under conditions that allow the duplex to enter the cell. Inparticular examples, the duplex is administered ex vivo or in vitro to acell, or administered directly to a subject. In another example, ansiRNA is part of a vector, and the vector administered ex vivo or invitro to a cell, or administered directly to a subject. In one example,the vector is the pSilencer™ 4.1-CMV vector (Ambion, Austin, Tex.).

Antisense Methods

Antisense oligonucleotides can be designed and generated using methodsknown in the art. For example, a sNRF genomic or mRNA sequence isexamined (such as nucleotides 1-215 of SEQ ID NO: 37). Regions of thesequence containing multiple repeats, such as TTTTTTTT, are not asdesirable because they will lack specificity. Several different regionscan be chosen. Of those, antisense oligonucleotides are selected by thefollowing characteristics: those having the best conformation insolution; those optimized for hybridization characteristics; and thosehaving less potential to form secondary structures. Antisense moleculeshaving a propensity to generate secondary structures are less desirable.

An antisense molecule that recognizes sNRF includes a sequencecomplementary to at least a portion of a sNRF RNA transcript. However,absolute complementarity is not required. An antisense sequence can becomplementary to at least a portion of an RNA, meaning a sequence havingsufficient complementarily to be able to hybridize with the RNA, forminga stable duplex; in the case of double-stranded antisense nucleic acids,a single strand of the duplex DNA may thus be tested, or triplexformation can be assayed. The ability to hybridize depends on the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

In a particular example, an antisense molecule that is specific for sNRFincludes at least 80% sequence identity, such as at least 90% sequenceidentity, to at least a fragment of a sNRF gene sequence, such asnucleotides 1-215 of SEQ ID NO: 37. In one example, the relative abilityof an antisense molecule to bind to its complementary nucleic acidsequence is compared by determining the T_(m) of a hybridization complexof the antisense molecule and its complementary strand. The higher theT_(m) the greater the strength of the binding of the hybridized strands.

Plasmids or vectors including antisense sequences that recognize sNRFcan be generated using standard methods. For example, cDNA fragments orvariants coding for a sNRF protein involved in infection are PCRamplified, for example using Pfu DNA polymerase (Stratagene). Theresulting sequence is cloned in antisense orientation a vector, such aspcDNA vectors (InVitrogen, Carlsbad, Calif.). The nucleotide sequenceand orientation of the insert can be confirmed by sequencing using aSequenase kit (Amersham Pharmacia Biotech). Such vectors can beadministered to a cell in a therapeutic amount, such as administered toa subject, to decrease one or more symptoms of heart failure.

Generally, the term “antisense” refers to a nucleic acid moleculecapable of hybridizing to a portion of a sNRF RNA sequence (such asmRNA) by virtue of some sequence complementarity. The antisense nucleicacids disclosed herein can be oligonucleotides that are double-strandedor single-stranded, RNA or DNA or a modification or derivative thereof,which can be directly administered to a cell (for example byadministering the antisense molecule to the subject), or which can beproduced intracellularly by transcription of exogenous, introducedsequences (for example by administering to the subject a vector thatincludes the antisense molecule under control of a promoter).

Antisense nucleic acids are polynucleotides, for example nucleic acidmolecules that are at least 6 nucleotides in length, at least 10nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least40 nucleotides, at least 100 nucleotides, at least 200 nucleotides, suchas 6 to 100 nucleotides. However, antisense molecules can be muchlonger. In particular examples, the nucleotide is modified at one ormore base moiety, sugar moiety, or phosphate backbone (or combinationsthereof), and can include other appending groups such as peptides, oragents facilitating transport across the cell membrane (Letsinger etal., Proc. Natl. Acad. Sci. USA 1989, 86:6553-6; Lemaitre et al., Proc.Natl. Acad. Sci. USA 1987, 84:648-52; WO 88/09810) or blood-brainbarrier (WO 89/10134), hybridization triggered cleavage agents (Krol etal., BioTechniques 1988, 6:958-76) or intercalating agents (Zon, Pharm.Res. 5:539-49, 1988).

Examples of modified base moieties include, but are not limited to:5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N-6-sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

Examples of modified sugar moieties include, but are not limited to:arabinose, 2-fluoroarabinose, xylose, and hexose, or a modifiedcomponent of the phosphate backbone, such as phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or aformacetal or analog thereof.

In a particular example, an antisense molecule is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625-41, 1987). The oligonucleotide can beconjugated to another molecule, such as a peptide, hybridizationtriggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent. Oligonucleotides can include atargeting moiety that enhances uptake of the molecule by host cells. Thetargeting moiety can be a specific binding molecule, such as an antibodyor fragment thereof that recognizes a molecule present on the surface ofthe host cell.

In a specific example, antisense molecules that recognize a sNRF nucleicacid molecule, include a catalytic RNA or a ribozyme (for example see WO90/11364; WO 95/06764; and Sarver et al., Science 247:1222-5, 1990).Conjugates of antisense with a metal complex, such as terpyridylCu (II),capable of mediating mRNA hydrolysis, are described in Bashkin et al.(Appl. Biochem Biotechnol. 54:43-56, 1995). In one example, theantisense nucleotide is a 2′-0-methylribonucleotide (Inoue et al., Nucl.Acids Res. 15:6131-48, 1987), or a chimeric RNA-DNA analogue (Inoue etal., FEBS Lett. 215:327-30, 1987).

Ribozymes

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by an endonucleolytic cleavage. Methods of using ribozymesto decrease or inhibit RNA expression are known in the art (for examplesee Kashani-Sabet, J. Investig. Dermatol. Symp. Proc., 7:76-78, 2002).

Ribozyme molecules include one or more sequences complementary to a sNRFgenomic or mRNA sequence (such as complementary to nucleotides 1-215 ofSEQ ID NO: 37) and include the well-known catalytic sequence responsiblefor mRNA cleavage (see U.S. Pat. No. 5,093,246, herein incorporated byreference).

Methods of designing and generating ribozyme molecules are known in theart. Briefly, specific ribozyme cleavage sites within a sNRF RNA target(such as nucleotides 1-215 of SEQ ID NO: 37) are identified by scanningthe RNA sequence for ribozyme cleavage sites that include: GUA, GUU andGUC. Once identified, RNA sequences of between 15 and 50 ribonucleotides(such as at least 20 ribonucleotides, at least 40 ribonucleotides, forexample 15-40, or 20-30 ribonucleotides) corresponding to the region ofsNRF containing the cleavage site can be evaluated for predictedstructural features, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatetargets cam also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

In particular examples, ribozymes are administered directly to asubject. In another example, a ribozyme is encoded on an expressionvector, from which the ribozyme is synthesized in a cell (as in WO9523225, and Beigelman et al. Nucl. Acids Res. 1995, 23:4434-42). Such acell or the vector can be administered to a subject.

In a specific example, a vector that contains a ribozyme gene directedagainst sNRF, placed behind a promoter (such as an inducible promoter),is transfected into the cells of a subject, for example a subject havingor susceptible to developing heart failure. Expression of this vector ina cell will decrease or inhibit sNRF RNA expression in the cell. In oneexample, the vector is the pSilencer™ 4.1-CMV vector (Ambion).

In a particular example, a vector includes self-cleaving tandemribozymes (for example, 5 ribozymes encoded on a single RNA transcript).Once the tandem hammerhead transcript is synthesized, it recognizesribozyme cleavage sites in cis within the newly synthesized transcriptidentical to the ribozyme target site on the cell's endogenouslyexpressed mRNA. The tandem ribozyme then cleaves itself, liberating freeribozymes to cleave the target mRNA in the cell.

microRNAs

MicroRNAs (miR5) that recognize a sNRF mRNA, can be used to decrease theamount of such mRNAs in a cell. miRNAs silence at thepost-transcriptional level by virtue of their sequence complementarityto target mRNAs. In particular examples, miRs are about 18-26nucleotides in length, such as 21-26 nucleotides, such as at least 18nucleotides. Animal miRNAs are generally thought to recognize their mRNAtargets by incomplete base-pairing, leading to translational inhibitionof the target.

Methods of generating or identifying microRNAs are known in the art (forexample see Lagos-Quintana et al., Science, 294:853-8, 2001;Lagos-Quintana et al., Curr. Biol. 12:735-9, 2002; and Lagos-Quintana etal., RNA 9:175-9, 2003).

Triple Helix Molecules

Nucleic acid molecules used in triplex helix formation for sNRF areideally single stranded and composed of deoxynucleotides. The basecomposition of these oligonucleotides is designed to promote triplehelix formation via Hoogsteen base pairing rules, which generallyrequire sizeable stretches of either purines or pyrimidines to bepresent on one strand of a duplex. Nucleotide sequences can bepyrimidine-based, which will result in TAT and CGC+ triplets across thethree associated strands of the resulting triple helix. Thepyrimidine-rich molecules provide base complementarity to a purine-richregion of a single strand of the duplex in a parallel orientation tothat strand. In addition, nucleic acid molecules can be chosen that arepurine-rich, for example contain a stretch of guanidine residues. Thesemolecules will form a triple helix with a DNA duplex that is rich in GCpairs, in which the majority of the purine residues are located on asingle strand of the targeted duplex, resulting in GGC triplets acrossthe three strands in the triplex.

Alternatively, sNRF sequences targeted for triple helix formation areincreased by creating “switchback” nucleic acid molecule. Switchbackmolecules are synthesized in an alternating 5′-3′,3′-5′ manner, suchthat they base pair with one strand of a duplex first and then theother, eliminating the necessity for a sizeable stretch of eitherpurines or pyrimidines to be present on one strand of a duplex.

sNRF inhibitory molecules shown to be effective in vitro can be furthertested in vivo, for example in a laboratory animal model for heartfailure (see Example 16).

Example 16 In vivo Testing of Agents

This example describes methods that can be used to test agents describedin Example 14 or 15, for their ability to increase the biologicalactivity of NPR (such as NPRA) in vivo, to decrease growth factordeleterious effects, or both, for example by decreasing the biologicalactivity of sNRF. Although particular methods are provided, one skilledin the art that other methods can be used, such as different animals,different modes of administration, and so forth.

The ability of an agent, such as those identified using the methodsprovided in Example 14 or the sNRF inhibitors disclosed in Example 15,or combinations thereof, can be assessed in animal models. For example,an animal model of cardiovascular disease can be administered theselected agent, and the effects on cardiovascular disease determined.

Such animal models are known in the art. For example, Kai et al.(Hypertens Res. 28:483-90, 2005) disclose Wistar rats with a suprarenalaortic constriction (AC) can be used as a model of cardiac hypertrophy,and Okuda et al. (Hypertens Res. 28:431-8, 2005) disclose thatadministration of isoproterenol (ISP) at 300 mg/kg into male rats canproduce progressive heart failure. In addition, Patel et al. (Am. J.Physiol. Heart Circ. Physiol. 289:H777-84, 2005, herein incorporated byreference) disclose transgenic mice that have cardiomyocyte-specificexpression of a dominant negative mutation (HCAT C893A) in the NPRAreceptor but no expression of the mutant receptor in the CF population.Thus the receptors in CFs in this model are likely to have dysregulatedNPRA. Any of these animal models can be used to analyze the effects ofone or more test agents on their ability to increase the activity of NPR(such as NPRA), to decrease growth factor deleterious effects, or both,for example to treat cardiovascular disease. Animals of any species,including, but not limited to, mice, rats, rabbits, guinea pigs, pigs,micro-pigs, goats, and non-human primates, such as baboons, monkeys, andchimpanzees, can be used.

In addition, such animal models can be used to determine the LD₅₀ andthe ED₅₀ in animal subjects, and such data can be used to determine thein vivo efficacy of potential agents.

The mammal is administered one or more agents identified in the examplesabove, alone or in combination with other therapeutic agents. The amountof agent administered, and the mode of administration, can be determinedby skilled practitioners. In some examples, several different doses ofthe potential therapeutic agent can be administered via severaldifferent routes to different test subjects, to identify optimal doseranges and modes of administration. Subsequent to the treatment, animalsare observed for decreases in signs and symptoms associated withcardiovascular disease, such as disease that results fromdesensitization of NPR for NPs (for example heart failure). Animalsubjects can be anesthetized prior to such treatments and evaluations.

In a specific example, sNRF siRNA (0.1-10 mg/kg), such as one or more ofthe exemplary siRNA duplex molecules shown in SEQ ID NOS: 14, 16, 18,20, 22, 24, 26, 28, 30, or 32 (or a duplex hybridized to itscomplement), is administered to Wistar rats with a suprarenal aorticconstriction (AC) via intravenous administration. In some examples, thesiRNA is administered before the rat receives the suprarenal AC, todetermine if the siRNA can reduce the effects of suprarenal AC (forexample to function as a prophylactic).

In animal models, NPR function can assessed by measuring the circulatinglevels or urinary levels of cGMP in response to NP infusion. A typicalbioassay of NPR function is to measure changes in forearm vascularresistance in response to NP infusion. Other common bioassays can be theurine output response to NP infusion. In animal models an agent mayincrease the activity of NPR (such as desensitized NPR) when treatmentof the animal with the agent results in the typical response of theanimal as if the NPR is not desensitized. Such an animal might havenormalized blood pressure, increased urine output, suppression ofnumerous neuroendocrine markers of heart failure such as angiotensin,aldosterone, endothelin, renin, the sympathetic nervous system, or othergrowth factors, combing programmed cell death (apoptosis), decreasedcardiac fibrosis, decrease in cardiac filling pressures, improvement ofcardiac output, and in general diminution of the typical signs andsymptoms of cardiovascular disease.

A decrease in the symptoms associated with cardiovascular disease thatresults from desensitization of NPR for NPs, in the presence of theagent provides evidence that the agent is a therapeutic agent that canbe used to increase the biological activity of NPR or decrease growthfactor (such as FGF and TGFβ1) deleterious effects, and therefore can beused to treat cardiovascular disease.

Example 17 Identification of Subjects that could Benefit from Therapy

This example describes methods that can be used to screen a subject todetermine if they have cardiovascular disease, or have an increased riskof developing cardiovascular disease. One skilled in the art willappreciate that other methods can be used, and that these methods aremerely exemplary to provide guidance as to particular examples that canbe used.

Generally, the method includes (if not already known) performing one ormore diagnostic assays that are indicative of cardiovascular disease,such as heart failure. The most straight forward method of definingthose with cardiovascular disease is define subjects based on the NewYork Heart classification of heart failure. Physical examination willreveal typical signs and symptoms of heart failure. Measurement of NPswill reveal patients with cardiovascular disease who are likely tobenefit from the presence of agents that promote NPR PKG interaction,reduce deleterious growth factor effects, for example agents thatdecrease expression or activity of sNRF. Those with high circulatinglevels of NPs (>50-100 pg/ml) or increased serum cGMP (>8 pg/ml) maybenefit from treatment.

In one example, the method includes analyzing a sample obtained from thesubject, such as a blood sample (or fraction thereof). Serum or otherblood fractions can be prepared in the conventional manner. For example,the levels of cGMP, ANP, BNP, triglycerides, cholesterol, can bedetermined using routine methods. Elevated levels of cGMP, ANP, BNP, orC-reactive protein, can indicate the presence of cardiovascular disease,while elevated levels of triglycerides or cholesterol can indicate thepresence of cardiovascular disease or an increased risk of developingcardiovascular disease in the future.

In a particular example, the subject is subjected to a stress test.

In another example, the subject is subjected to an angiogram.

Subjects found to have cardiovascular disease, or have an increased riskof developing cardiovascular disease, can be administered the agentsidentified using the methods disclosed herein (see Example 18).

Example 18 Treatment of Subjects

This example describes methods that can be used to treat a subjecthaving a heart disease (such as heart failure), or having an increasedrisk of developing heart disease in the future, by administration of oneor more of the agents that decrease the biological activity of sNRF(such as siRNAs, see Example 15), or one or more agents identified usingthe methods disclosed herein (for example see Example 14). For example,the disclosed methods can be used to increase the sensitivity of an NPRfor NP ligands (such as ANP) or decrease or inhibit the deleteriouseffects of growth factor action, such as the effects of TGFβ1 or FGF,for example to treat or reduce the symptoms of cardiovascular disease.For example, the disclosed methods can be used to. Such a therapy can beused alone, or in combination with other therapies (such asadministration of a statin or anti-coagulant).

In particular examples, the method includes screening a subject havingor thought to have (or be an increased risk of developing)cardiovascular disease, to identify those subjects that can benefit fromadministration of the therapeutic agents disclosed herein. As describedin Example 17, subjects of an unknown cardiovascular disease status canbe examined to determine if they have cardiovascular disease, or have anincreased risk of developing cardiovascular disease. Subjects found to(or known to) have cardiovascular disease, or have an increased risk ofdeveloping cardiovascular disease, are selected to receive one or moreagents disclosed herein.

As described in Example 14, screening methods can be used to identifyagents that increase the activity of NPR or decrease growth factor (suchas FGF and TGFβ1) deleterious effects. These agents, such as antibodies,peptides, nucleic acid molecules, organic or inorganic compounds, aswell as the RNAi molecules described in Example 15, can be administeredto a subject in a therapeutically effective amount to treat heartdisease. Therefore, those agents can be administered to a subject havingor at risk for developing cardiovascular disease at a therapeuticallyeffective dose, thereby relieving the symptoms associated withcardiovascular disease or to prevent cardiovascular disease. Such agentscan also be administered with other therapeutic agents, such as statinsor anti-coagulants. By increasing the activity of NPRA/B, decreasing theactivity of sNRF, decreasing growth factor (such as FGF and TGFβ1)deleterious effects, or combinations thereof, for example by increasingthe sensitivity of NPRA/B for NP (such as ANP), the subject can takeadvantage of the naturally high levels of NPs that occur duringcardiovascular disease (such as heart failure) and thereby capitalize onthe “natural defenses” provided by NPs against cardiovascular disease.

The subject can be administered a therapeutic amount of one or more ofthe agents identified using the methods disclosed herein. The agents canbe administered at doses of 1 μg/kg body weight to about 1 mg/kg bodyweight per dose, such as 1 μg/kg body weight −100 μg/kg body weight perdose, 100 μg/kg body weight −500 μg/kg body weight per dose, or 500μg/kg body weight −1000 μg/kg body weight per dose. However, theparticular dose can be determined by a skilled clinician. The agent canbe administered in several doses, for example continuously, daily,weekly, or monthly.

Increasing the activity of NPRA/B need not result in 100% of native ofNPRA/B function (for example 100% NPR resensitization), nor 100%inhibition of deleterious growth factor actions, nor treatment of 100%of the symptoms associated with the cardiovascular disease.

Example 15 describes molecules that decrease the biological activity ofsNRF (such as decrease or inhibit the expression of sNRF mRNA). Suchagents can be administered to a subject in a therapeutically effectiveamount that decreases the biological activity of sNRF. When the activityof sNRF is decreased, for example by prematurely downregulating theirprotein or nucleic acid molecule levels, a reduction in one or moresymptoms associated with heart failure is achieved. For example,antisense oligonucleotides, ribozymes, miRs, siRNA, and triple helixmolecules that recognize a nucleic acid that encodes sNRF can thereforebe used to disrupt cellular expression of sNRF. The disclosed antisense,ribozyme, triple helix, miRs, and siRNA molecules (for example at aconcentration of 1-100 mg nucleic acid molecule/kg of subject, such as1-10 mg/kg, 1-20 mg/kg, 10-50 mg/kg, or 40-80 mg/kg) can be administeredto a subject alone, or in combination with other agents, such as apharmaceutical carrier, other therapeutic agents (such as agents used totreat heart disease, such as statins and anti-coagulants), orcombinations thereof. In one example, the subject is a mammal, such asmice, non-human primates, and humans.

In one example, an siRNA, ribozyme, triple helix, miR, or antisensemolecule specific for sNRF is part of a vector, and the vectoradministered ex vivo or in vitro to a cell, or administered directly toa subject. For example, a U6 promotor that controls the expression of 21nucleotides, followed by a stem-loop of 8 bp, followed by an additionalcomplementary 21 bp that anneals to the first 21 nucleotidestranscribed. The 21 nucleotide sequences are the siRNA that recognizesNRF (such as SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32).Transcription is halted using a stretch of 5 T's in the plasmidimmediately downstream of the last desired transcribed nucleotide. Inanother example, the vector is the pSilencer™ 4.1-CMV vector (Ambion).

In particular examples, a subject susceptible to or suffering from heartfailure, wherein decreased symptoms associated with heart failure isdesired, is treated with a therapeutically effective amount ofantisense, ribozyme, triple helix, miR, or siRNA molecule (orcombinations thereof) that recognizes a sNRF nucleic acid sequence (forexample one that is complementary to at least 20 contiguous nucleotides,such as at least 21 contiguous nucleotides, of nucleotides 1-215 of SEQID NO: 37). Similarly, other agents, such as an agent that specificallyrecognizes and interacts with (such as binds to) a sNRF protein, therebydecreasing the ability of the sNRF protein to interfere with PKG-NPRbinding, decreasing growth factor deleterious effects, or both, can alsobe used to treat heart failure.

After the agent has produced an effect (decreased symptoms associatedwith heart failure), for example after 24-48 hours, the subject can bemonitored for symptoms associated with heart failure.

The mode of administration can be any used in the art, such as thosedescribed in Example 19. The amount of agent administered to the subjectcan be determined by a clinician, and may depend on the particularsubject treated. Specific exemplary amounts are provided herein (but thedisclosure is not limited to such doses).

Example 19 Pharmaceutical Compositions and Modes of Administration

This example provides methods and pharmaceutical compositions that canbe used to administer a therapeutic agent disclosed herein (alone or incombination with other therapeutic agents), such as those that canincrease the biological activity of NPRA or NPRB, decrease growth factordeleterious effects, or combinations thereof. Such agents include thoseidentified using the methods described in Example 14 and the RNAimolecules described in Example 15.

Administration of such compositions to a subject can begin whenevertreatment of symptoms associated with cardiovascular disease associatedwith decreased NPRA biological activity (such as desensitization of NPRAto ANP) or associated with insufficient sNRF-induced potentiation ofdeleterious growth factor effects, is desired. While compositions thatinclude such therapeutic agents are typically be used to treat humansubjects, they can also be used to treat other vertebrates such as otherprimates, farm animals such as swine, cattle and poultry, and sportanimals and pets such as horses, dogs and cats.

The pharmaceutical compositions that include a therapeutic agent (forexample see Examples 14 and 15) that can increase the biologicalactivity of NPRA or NPRB, decrease the biological activity of sNRF,reduce sNRF-induced potentiation of deleterious growth factor effects,or combinations thereof, can be formulated in unit dosage form, suitablefor individual administration of precise dosages. A therapeuticallyeffective amount of such agents can be administered in a single dose, orin multiple doses, for example daily, during a course of treatment.Compositions that include such agents can be administered whenever theeffect (such as decreased symptoms of cardiovascular disease) isdesired. A time-release formulation can also be utilized.

A therapeutically effective amount of a composition that includes thetherapeutic agent can be administered as a single pulse dose, as a bolusdose, or as pulse doses administered over time. In specific,non-limiting examples, pulse doses of compositions that include theagent are administered during the course of a day, during the course ofa week, or during the course of a month.

The therapeutically effective amount of a composition including thetherapeutic agent can depend on the molecule utilized, the subject beingtreated, the severity and type of the affliction, and the manner ofadministration, and should be decided according to the judgment of thepractitioner and each subject's circumstances. Therapeutically effectiveamounts of compositions that include the therapeutic agent are thosethat restore function to a desensitized NPRA or NPRB by an amount thatdecreases one or more symptoms of cardiovascular disease, an amount thatincreases the sensitivity of NPRA or NPRB for ANP or BNP (for example anincrease of at least 20% or at least 50%), those that reducesNRF-induced potentiation of deleterious growth factor effects, orcombinations thereof. In vitro assays can be employed to identifyoptimal dosage ranges. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.For example, a therapeutically effective amount of the therapeutic agentcan vary from about 0.001 μg per kilogram (kg) body weight to about 20mg per kg body weight, such as about 1 μg to about 5 mg per kg bodyweight, about 2 μg to about 0.5 mg per kg body weight, about 1 μg toabout 50 μg per kg body weight, about 10 μg to about 100 μg per kg bodyweight, or about 5 μg to about 1 mg per kg body weight. The exact doseis readily determined by one of skill in the art based on the potency ofthe specific compound utilized, the age, weight, sex and physiologicalcondition of the subject.

The compositions or pharmaceutical compositions can be administered byany route, including intravenous, intraperitoneal, subcutaneous,sublingual, transdermal, intramuscular, oral, topical, transmucosal,vaginal, nasal, rectal, by pulmonary inhalation, or combinationsthereof. Compositions useful in the disclosure may conveniently beprovided in the form of formulations suitable for parenteral (includingintravenous, intramuscular and subcutaneous), nasal, topical, or oraladministration.

In some examples, compositions that include the therapeutic agent areadministered in combination with (such as before, during, or following)a therapeutically effective amount of one or more other therapeuticagents, in a single composition or solution for administration together.In other cases, it may be more advantageous to administer the additionalagent separately from the therapeutic agent identified using the methodsdisclosed herein. Compositions that include the therapeutic agent can beadministered simultaneously with the additional agent(s), oradministered sequentially. In one example, the other therapeutic agentis a statin, an anti-coagulant, or other agents that alleviate symptomsassociated with cardiovascular disease (or risk of developingcardiovascular disease).

Therapeutic compositions can be provided as parenteral compositions,such as for injection or infusion. Such compositions are formulatedgenerally by mixing the identified therapeutic agent at the desireddegree of purity, in a unit dosage injectable form (solution,suspension, or emulsion), with a pharmaceutically acceptable carrier,for example one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. In addition, a the identified therapeutic agent can besuspended in an aqueous carrier, for example, in an isotonic buffersolution at a pH of about 3.0 to about 8.0, such as at a pH of about 3.5to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers includesodium citrate-citric acid and sodium phosphate-phosphoric acid, andsodium acetate/acetic acid buffers. The active ingredient, optionallytogether with excipients, can also be in the form of a lyophilisate andcan be made into a solution prior to parenteral administration by theaddition of suitable solvents. Solutions such as those that are used,for example, for parenteral administration can also be used as infusionsolutions.

A form of repository or “depot” slow release preparation can be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection or delivery. Such long acting formulations can be administeredby implantation (for example subcutaneously or intramuscularly) or byintramuscular injection. The compounds can be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions that include the identified therapeuticagent as an active ingredient can be formulated with an appropriatesolid or liquid carrier, depending upon the particular mode ofadministration chosen. The product can be shaped into the desiredformulation. In one example, the carrier is a parenteral carrier, suchas a solution that is isotonic with the blood of the recipient. Examplesof such carrier vehicles include water, saline, Ringer's solution,glycerol and dextrose solution. Non-aqueous vehicles such as fixed oilsand ethyl oleate are also useful herein, as well as liposomes. Othercarriers include, but are not limited to: fillers, such as sugars, forexample lactose, saccharose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example tricalcium phosphateor calcium hydrogen phosphate, also binders, such as starches, forexample corn, wheat, rice or potato starch, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose orpolyvinylpyffolidone, and/or, if desired, disintegrators, such as theabove-mentioned starches, also carboxymethyl starch, cross-linkedpolyvinylpyrrolidone, alginic acid or a salt thereof, such as sodiumalginate. Additional pharmaceutically acceptable carriers and theirformulation are described in standard formulation treatises, such asRemington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y.J. and Hanson, M. A., Journal of Parenteral Science and Technology,Technical Report No. 10, Supp. 42:2 S, 1988.

If desired, the disclosed pharmaceutical compositions can also containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate. Excipients that canbe included in the disclosed compositions include flow conditioners andlubricants, for example silicic acid, talc, stearic acid or saltsthereof, such as magnesium or calcium stearate, and/or polyethyleneglycol, or derivatives thereof.

Compositions including the identified therapeutic agent can beadministered by sustained-release systems. Suitable examples ofsustained-release systems include suitable polymeric materials (such as,semi-permeable polymer matrices in the form of shaped articles, forexample films, or mirocapsules), suitable hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, andsparingly soluble derivatives (such as, for example, a sparingly solublesalt). Sustained-release compositions can be administered orally,parenterally, intracistemally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), or as an oral,otic, or nasal spray. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556, 1983,poly(2-hydroxyethyl methacrylate)); (Langer et al., J. Biomed. Mater.Res.15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982, ethylenevinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid(EP 133,988).

Sustained-release compositions include liposomes containing theidentified therapeutic agent (see generally, Langer, Science249:1527-1533, 1990; Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-65, 1989). Liposomes containing theidentified therapeutic agent thereof can be prepared by known methods:DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A. 82:3688-92,1985; Hwang et al., Proc. Natl. Acad. Sci. U.S.A. 77:4030-4034, 1980; EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese PatentApplication No. 83-118008; U.S. Pat. No. 4,485,045, U.S. Pat. No.4,544,545; and EP 102,324.

Preparations for administration can be suitably formulated to givecontrolled release of a therapeutic agent disclosed herein. For example,the pharmaceutical compositions can be in the form of particlescomprising a biodegradable polymer or a polysaccharide jellifying orbioadhesive polymer, an amphiphilic polymer, an agent modifying theinterface properties of the particles and a pharmacologically activesubstance. These compositions exhibit certain biocompatibility featuresthat allow a controlled release of the active substance. See U.S. Pat.No. 5,700,486.

Compositions that include the identified therapeutic agent can bedelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudeket al., N. Engl. J. Med. 321:574, 1989) or by continuous subcutaneousinfusions, for example, using a mini-pump. An intravenous bag solutioncan also be employed. One factor in selecting an appropriate dose is theresult obtained, as measured by the methods disclosed here, as aredeemed appropriate by the practitioner. Other controlled release systemsare discussed in Langer (Science 249:1527-33, 1990).

In one example, the pump is implanted (for example see U.S. Pat. Nos.6,436,091; 5,939,380; and 5,993,414). Implantable drug infusion devicesare used to provide patients with a constant and long-term dosage orinfusion of a drug or any other therapeutic agent. Such device can becategorized as either active or passive.

Active drug or programmable infusion devices feature a pump or ametering system to deliver the agent into the patient's system. Anexample of such an active infusion device currently available is theMedtronic SynchroMed™ programmable pump. Passive infusion devices, incontrast, do not feature a pump, but rather rely upon a pressurized drugreservoir to deliver the agent of interest. An example of such a deviceincludes the Medtronic IsoMed™.

For oral administration, the pharmaceutical compositions can take theform of, for example, powders, pills, tablets, or capsules, prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (such as pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such aslactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (such as magnesium stearate, talc or silica); disintegrants(such as potato starch or sodium starch glycolate); or wetting agents(such as sodium lauryl sulphate). The tablets can be coated by methodswell known in the art.

For administration by inhalation, the compounds for use according to thepresent disclosure can be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of for example gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

For inhalation, the composition of the present disclosure can also beadministered as an aerosol or as a dispersion in a carrier. In onespecific, non-limiting example, the identified therapeutic agent (aloneor in combination with other therapeutic agents or pharmaceuticallyacceptable carriers), is administered as an aerosol from a conventionalvalve, such as, but not limited to, a metered dose valve, through anaerosol adapter also known as an actuator. A suitable fluid carrier canbe also included in the formulation, such as, but not limited to, air, ahydrocarbon, such as n-butane, propane, isopentane, amongst others, or apropellant, such as, but not limited to a fluorocarbon. Optionally, astabilizer is also included, or porous particles for deep lung deliveryare included (for example, see U.S. Pat. No. 6,447,743).

In the disclosed methods of treating cardiac disorders that result fromdecreased sensitivity of NPR for NP, the method includes administeringto a subject (such as a subject having cardiovascular disease) atherapeutically effective amount of a therapeutic agent identified usingthe methods disclosed herein. Such agents can be administered in asingle or divided dose. In particular examples, suitable single ordivided doses include, but are not limited to about 0.01, 0.1, 0.5, 1,3, 5, 10, 15, 30, or 50 μg of agent/kg of subject/day.

The disclosure also provides a pharmaceutical pack or kit including oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. Instructions for useof the composition can also be included.

The disclosure provides compositions that include one or more of theidentified therapeutic agents using the methods described in Example 14,one or more of the RNAi molecules described in Example 15, orcombinations thereof, for example a composition that includes at least50%, for example at least 90%, of the therapeutic agent in thecomposition. Such compositions are useful as therapeutic agents whenconstituted as pharmaceutical compositions with the appropriate carriersor diluents.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated examples are only examples of the invention and should notbe taken as limiting the scope of the invention. Rather, the scope ofthe invention is defined by the following claims. We therefore claim asour invention all that comes within the scope and spirit of theseclaims.

1. A method of treating cardiovascular disease, comprising significantlydecreasing sNRF activity in a cardiac cell, wherein significantlydecreasing sNRF activity treats the cardiovascular disease.
 2. Themethod of claim 1, wherein significantly decreasing sNRF activitycomprises decreasing expression of an mRNA encoding sNRF in the cell,increasing sensitivity of a natruiuretic peptide receptor (NRP) in thecell for NP, decreasing deleterious growth factor effects, orcombinations thereof.
 3. The method of claim 2, wherein significantlydecreasing expression of the mRNA encoding sNRF comprises contacting themRNA with an antisense RNA, triple helix molecule, ribozyme, microRNA,or siRNA that recognizes the sNRF mRNA.
 4. The method of claim 3,wherein the antisense RNA, triple helix molecule, ribozyme, microRNA, orsiRNA that recognizes the sNRF mRNA is complementart to at least 20contiguous of nucleotides 1-215 of SEQ ID NO:
 37. 5. The method of claim4, wherein decreasing expression of mRNA encoding sNRF comprisescontacting the mRNA with one or more of SEQ ID NOS: 14, 16, 18, 20, 22,24, 26, 28, 30, or
 32. 6. The method of claim 4, wherein decreasingexpression of mRNA encoding sNRF comprises contacting the mRNA with oneor more duplexes formed by SEQ ID NOS: 13 and 14, 15 and 16, 17 and 18,19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, or 31and
 32. 7. The method of claim 1, wherein sNRF is encoded by a nucleicacid molecule comprising at least 95% sequence identity to any of SEQ IDNOS: 3, 5, 37, 39, or
 41. 8. The method of claim 7, wherein sNRF isencoded by a nucleic acid molecule consisting of any of SEQ ID NOS: 3,5, 37, 39 or
 41. 9. The method of claim 1, wherein the cardiac cell is ahuman cardiac cell.
 10. The method of claim 1, wherein the cardiac cellis present in a subject, and decreasing activity of sNRF in a cardiaccell comprises administering a therapeutically effective amount of anagent that decreases activity of sNRF in the cardiac cell to thesubject.
 11. A method of treating a subject having cardiovasculardisease, comprising: determining whether the subject has cardiovasculardisease or is at an increased risk for developing cardiovasculardisease; administering a therapeutically effective amount of an agentthat significantly decreases sNRF activity to a subject havingcardiovascular disease or having an increased risk for developingcardiovascular disease, which indicates that the subject can be treatedwith the agent, thereby treating the cardiovascular disease.
 12. Themethod of claim 1, wherein the cardiovascular disease is anginapectoris; arrhythmia; cardiac fibrosis, congenital cardiovasculardisease; coronary artery disease (CAD); dilated cardiomyopathy; heartattack (myocardial infarction); heart failure; hypertrophiccardiomyopathy; systemic hypertension from any cause, edematousdisorders caused by liver or renal disease, mitral regurgitation,myocardial tumors, myocarditis, rheumatic fever, Kawasaki disease,Takaysu arteritis, cor pulmonale, primary pulmonary hypertension,amyloidosis, hemachromatosis, toxic effects on the heart due topoisoning, Chaga's disease, heart transplantation, cardiac rejectionafter heart transplantation, cardiomyopathy of chachexia, arrhythmogenicright ventricular dysplasia, cardiomyopathy of pregnancy, MarfanSyndrome; Turner Syndrome; Loeys-Dietz Syndrome, familial biscuspidaortic valve, or any inherited disorder of the heart or vasculature, orcombinations thereof.
 13. The method of claim 11, wherein determiningwhether the subject has cardiovascular disease or is at an increasedrisk for developing cardiovascular disease comprises determining serumlevels of ANP, BNP, or cGMP, wherein a serum level of >50-100 pg/ml forANP, a serum level of 50-100 pgt/ml for BNP, or a serum level of >8pg/ml for cGMP, indicates that the subject has cardiovascular disease oris at an increased risk for developing cardiovascular disease.
 14. Themethod of claim 1, wherein the cardiovascular disease results from adesensitized NPRA or increased growth factor deleterious effects.
 15. Anisolated protein comprising the sequence of SEQ ID NO: 4, 6, 8, 38, 40or
 42. 16. An isolated nucleic acid molecule encoding the protein ofclaim
 15. 17. The isolated nucleic acid molecule of claim 16, whereinthe isolated nucleic acid molecule consists of SEQ ID NO: 3, 5, 7, 37,39, or 41.